KR102709830B1 - Perfusion manifold assembly - Google Patents

Abstract

A drop-to-drop connection method for placing a microfluidic device in fluid communication with a fluid source or another microfluidic device, such as but not limited to placing a microfluidic device in fluid communication with a perfusion manifold assembly, is described. A perfusion manifold assembly is described that allows perfusion of a microfluidic device, such as an organ on a chip microfluidic device including cells that mimic cells found in an organ within the body, releasably connected to the assembly such that fluid flows from a fluid reservoir to a port of the microfluidic device at a controllable flow rate, optionally without tubing.

Description

{PERFUSION MANIFOLD ASSEMBLY}

A perfusion manifold assembly is contemplated that allows perfusion of a microfluidic device, such as an organ on a chip microfluidic device comprising cells that mimic cells found in an organ of the body or that mimic at least one function of an organ, removably connected to the assembly such that fluid can flow from a fluid reservoir to a port of the microfluidic device at a controllable flow rate without tubing. A droplet-to-droplet connection is contemplated as one embodiment for placing the microfluidic device in fluid communication with a fluid source or with another microfluidic device, such as but not limited to, placing the microfluidic device in fluid communication with the perfusion manifold assembly.

Two-dimensional (2D) monolayer cell culture systems have been used in biological research for many years. The most common cell culture platform is a two-dimensional (2D) monolayer cell culture in a petri dish or flask. Although these 2D in vitro models are inexpensive compared to animal models and contribute to systematic, reproducible, quantitative studies of cell physiology (e.g., in drug discovery and development), the physiological relevance of information retrieved from in vitro studies to in vivo systems is often questionable. It is now widely accepted that three-dimensional (3D) cell culture matrices promote several biologically relevant functions that are not observed in 2D monolayer cell cultures. In other words, 2D cell culture systems do not accurately recapitulate the structure, function, and physiology of living tissues in vivo.

U.S. Patent No. 8,647,861 describes a microfluidic "organ-on-chip" device comprising living cells on a membrane in microchannels exposed to culture fluid at a predetermined flow rate. In contrast to static 2D cultures, the microchannels allow for perfusion of cell culture medium through the cell culture during in vitro studies, thus providing a physical environment more similar to that in vivo. Briefly, an infusion port allows for the introduction of cell culture medium into the cell-containing microfluidic channels or chambers, thereby delivering nutrients and oxygen to the cells. An exhaust port then allows for the discharge of the remaining medium as well as harmful metabolic byproducts.

Although these microfluidic devices represent an improvement over traditional static tissue culture models, the small size, scale, and interfaces of these devices make fluid handling difficult. A method is needed to control perfusion in these devices in such a way that the fluid pressure generates a flow rate that applies the desired fluid shear stress to the living cells. Ideally, such a solution should provide a simple user workflow.

Summary of the invention

The present invention contemplates a number of devices, both individually and in combination. The present invention contemplates a perfusion manifold assembly (also referred to as a cartridge, pod, or perfusion disposable, whether or not there is any requirement or intent as to the arrangement of components) containing one or more microfluidic devices, such as an "organ-on-a-chip" microfluidic device (or simply a "microfluidic chip"), that comprises cells that mimic at least one function of an organ in the body and optionally permits perfusion and optionally mechanical actuation of the microfluidic device without tubing. The present invention contemplates a number of embodiments of a perfusion manifold assembly. However, the present invention is not intended to be limited to these embodiments. For example, the present invention contemplates combining features from different embodiments (as discussed below). The present invention also contemplates removing features from embodiments (as discussed below). Additionally, the present invention contemplates substituting features in embodiments (as discussed below).

A culture module is contemplated that allows perfusion and optionally mechanical actuation of one or more microfluidic devices, such as an organ-on-a-chip microfluidic device comprising cells that mimic at least one function of an organ in the body. In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or within the membrane and/or on the channels. In one embodiment, the culture module comprises a pressure manifold that allows perfusion of the microfluidic device, such as an "organ-on-a-chip" microfluidic device comprising cells that mimic cells in an organ in the body or that mimic at least one function of an organ, optionally in contact with a perfusion disposable and removably connected to said assembly such that fluid is allowed to flow from a fluid reservoir into a port of the microfluidic device, optionally without tubing, at a controllable flow rate. The perfusion disposable can be used separately from the culture module, and the microfluidic device or chip can be used separately from the perfusion disposable. In one embodiment, the present invention contemplates a pressure manifold (e.g., any one of the flow-through manifold assembly embodiments described herein) configured to be coupled with one or more microfluidic devices having an integrated valve capable of preventing gas leakage when not coupled with the microfluidic device.

A drop-to-drop connection arrangement is contemplated as one embodiment for positioning the microfluidic device in fluid communication with a fluid source or another microfluidic device, such as but not limited to positioning the microfluidic device in fluid communication with a perfusion disposable. In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or within the membrane and/or on the channels.

A pressure cover for pressurizing one or more reservoirs within a perfusion disposable or a perfusion manifold assembly (or other microfluidic device) is contemplated, wherein the pressure cover is movable or removably attached to the perfusion disposable or other microfluidic device to allow improved access to the member (e.g., the reservoir) from within. The pressure cover can be removed from the perfusion disposable, and the perfusion disposable can be used without the cover. In one embodiment, the perfusion disposable comprises a microfluidic chip, the chip comprising an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic chip comprises cells on or within the membrane and/or the channels.

Despite the limitations of conventional pressure regulators, a pressure control method is contemplated that allows control of flow rate (while perfusing cells). In one embodiment, rather than the pressure controllers (or actuators) of a culture module being "on" all the time (or at just one setting), they are switched in a pattern of "on" and "off" (or between two or more settings). Thus, the switching pattern can be selected such that the average value of the liquid working pressure in one or more reservoirs of the interlocked perfusion disposable (containing the microfluidic device or chip) corresponds to a desired value. In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or in the membrane and/or on the channels.

In one embodiment, the perfusion manifold assembly comprises i) a cover or lid configured to act as an upper portion of one or more fluid reservoirs, ii) one or more fluid reservoirs, iii) a capping layer disposed beneath said fluid reservoir(s), iv) a fluid backplane comprising a resistor, in fluid communication with said fluid reservoir(s) beneath said fluid reservoir(s), and v) a protruding member or skirt (for engaging a microfluidic device or a carrier containing a microfluidic device). As noted above, the cover or lid can be removed and the perfusion manifold assembly can still be used. In one embodiment, the assembly further comprises a fluid port disposed beneath the fluid backplane. In one embodiment, the capping layer caps the fluid backplane. Without being bound by any particular mechanism, it is believed that these resistors act to stabilize the flow of fluid from the reservoirs such that a steady flow can be delivered to the microfluidic device and/or they act to provide a means for converting reservoir pressure into a perfusion flow rate. In one embodiment, the lid is held over the reservoirs using a radial seal. This does not require any applied pressure to create a seal. In another embodiment, the cover is retained on the reservoir using one or more clips, screws or other retention mechanisms. In one embodiment, the protruding member or skirt is engaged with the microfluidic chip. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels.

In one embodiment, the perfusion manifold assembly comprises i) one or more fluid reservoirs, and ii) a fluid backplane in fluid communication with and beneath said fluid reservoir(s) and terminating in a port. In one embodiment, the fluid backplane comprises a resistor. In one embodiment, the perfusion manifold assembly further comprises iii) a protruding member or skirt. In one embodiment, the skirt comprises a guide mechanism (for engaging a microfluidic device or a carrier containing a microfluidic device). In one embodiment, the guide mechanism comprises a guide shaft, or a hole, groove, orifice or other cavity configured to receive a guide shaft. In one embodiment, the guide mechanism comprises a guide track (external or internal). In one embodiment, the guide track is a side track (for engaging a microfluidic device or a carrier). In one embodiment, the perfusion manifold assembly can further comprise a capping layer capping the fluid backplane. The embodiment can optionally further comprise a cover or lid. In one embodiment, the lid is held on the reservoir using a radial seal. This does not require applied pressure to create a seal. In another embodiment, the cover is held on the reservoir using one or more clips, screws or other retaining mechanisms. In one embodiment, the fluid port is on the lower side of the fluid backplane. In one embodiment, the protruding member or skirt is engaged with the microfluidic chip. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels.

In one embodiment, a perfusion manifold assembly comprises i) one or more fluid reservoirs, ii) a fluid backplane comprising a resistor in fluid communication with and beneath said fluid reservoir(s), and iii) a protruding member or skirt (for engaging a microfluidic device or a carrier containing a microfluidic device). The embodiment can further comprise a capping layer capping the fluid backplane. The embodiment can optionally further comprise a cover or lid. In one embodiment, the lid is retained on the reservoir using a radial seal. This does not require applied pressure to create a seal. In another embodiment, the lid is retained on the reservoir using one or more clips, screws or other retaining mechanisms. In one embodiment, the fluid port is at the bottom of the fluid backplane. In one embodiment, the protruding member or skirt is engaged with the microfluidic chip. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels.

In one embodiment, the perfusion manifold assembly comprises i) one or more fluid reservoirs, ii) a fluid backplane in fluid communication with and beneath said fluid reservoir(s), and iii) a capping layer capping the fluid backplane. In one embodiment, the fluid backplane comprises one or more resistors. In one embodiment, the assembly optionally further comprises iv) a protruding member or skirt (for engaging with a microfluidic device or a carrier containing a microfluidic device). The embodiment may optionally further comprise a cover or lid. In some embodiments, attachment of the microfluidic device to the perfusion disposable is via engagement with the skirt. However, in other embodiments, attachment is achieved directly with the assembly (without a skirt or other external extension). In one embodiment, the protruding member or skirt is engaged with the microfluidic chip. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or within the membrane and/or the channels.

In one embodiment, the present invention contemplates a perfusion manifold assembly comprising i) one or more fluid reservoirs, ii) a fluid backplane comprising fluid channels terminating in ports and fluid resistors, the fluid backplane being positioned beneath and in fluid communication with the fluid reservoirs, and iii) a protruding member or skirt having one or more side tracks. In one embodiment, the ports are positioned beneath the fluid backplane. In one embodiment, the one or more side tracks are configured to engage with a microfluidic device positioned on a microfluidic device carrier having one or more outer edges configured to slidably engage with the one or more side tracks. In one embodiment of the slidable engagement, the connection approach to the perfusion manifold comprises 1) a sliding motion, 2) a pivoting motion, and 3) a snap fit to provide alignment and fluid connection in a single motion. In the 1) sliding step, a chip (or other microfluidic device) is placed into the carrier which slides to align the fluid ports. 2) In the pivot step, the carrier and chip (or other microfluidic device) are pivoted until the port comes into contact with the fluid. 3) In the clip or snap fit step, the force necessary to provide a secure seal is provided. In one embodiment, the protruding member or skirt is engaged with the microfluidic chip. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels.

In one embodiment, the carrier has a cutout or "window" (e.g., a transparent window) for imaging the cells within the microfluidic chip (e.g., using a microscope). In one embodiment, the cutout or window (e.g., is transparent) corresponds to a perfusion disposable. In one embodiment, the microfluidic device comprises features in the carrier to eliminate the need for a separate substrate. In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or within the membrane and/or on the channels.

In one embodiment, the present invention contemplates a perfusion manifold assembly comprising i) one or more fluid reservoirs, ii) a fluid backplane comprising fluid channels and fluid resistors terminating in a protruding member or skirt and positioned beneath and in fluid communication with the fluid reservoirs, iii) a protruding member or skirt having one or more fluid ports and one or more side tracks. In one embodiment, the one or more side tracks are configured to engage with a microfluidic device positioned on a microfluidic device carrier having one or more outer edges configured to slidably engage with the one or more side tracks. In one embodiment of the slidable engagement, the connection approach to the perfusion manifold comprises 1) a sliding motion, 2) a pivoting motion, and 3) a snap fit to provide alignment and fluid connection in a single operation. In the 1) sliding step, a chip (or other microfluidic device) is placed into the carrier which slides to align the fluid ports. 2) In the pivot step, the carrier and chip (or other microfluidic device) are pivoted until the port comes into contact with the fluid. 3) In the clip or snap fit step, the force necessary to provide a secure seal is provided. In one embodiment, the microfluidic device includes features in the carrier to eliminate the need for a separate substrate. In one embodiment, the carrier has a cutout or "window" (e.g., a transparent window) for imaging (e.g., using a microscope). In one embodiment, there is a cutout or window (e.g., transparent) corresponding to a perfusion disposable (e.g., in the fluid layer). In one embodiment, the invention contemplates control of the focal plane position and alignment (flatness relative to the microscope stage) on which the chip is placed. It is desirable to minimize the working distance required for imaging (since longer working distances put greater strain on the objective). The invention is not intended to be limited by the imaging approach, and imaging can be upright (objective from above) or flipped (objective from below). While certain embodiments have cutouts or windows on only one side for a particular imaging modality (e.g., epifluorescence), in preferred embodiments the present invention contemplates cutouts or windows on both sides of the chip to enable transmitted light imaging. In one embodiment, the resistor comprises a tortuous channel. In one embodiment, the fluidic backplane is made of a cyclo olefin polymer (COP) (e.g., commercially available Zeonor 1420R) and comprises a linear fluidic channel in fluid communication with the tortuous channel, the linear channel terminating in one or more ports. In one embodiment, the skirt is made of polycarbonate (PC). In one embodiment, the assembly further comprises a cover for the fluid reservoir, the cover comprising a plurality of ports optionally associated with a filter. In some embodiments, the cover ports comprise through-holes, and the filter is positioned over corresponding holes in the gasket. In some embodiments, the cover comprises one or more channels passing through one or more of the ports (such that the ports are not simple through-holes). In one embodiment, the side track comprises a closed first end proximate the reservoir and an open second end distal from the reservoir, the open end comprising an angled slide for engaging the one or more outer edges of the microfluidic device carrier. In one embodiment, the side track comprises a linear region between the closed first end and the open second end. In one embodiment, a protruding member or skirt engages the microfluidic chip. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or within the membrane and/or on the channels.

The present invention also contemplates a system comprising a perfusion manifold assembly. In one embodiment, the present invention contemplates a system comprising: a) a perfusion manifold assembly comprising i) one or more fluid reservoirs, ii) a fluid backplane positioned below and in fluid communication with the fluid reservoirs, and iii) a skirt or other protruding member; and b) a microfluidic device or chip that is engaged with the perfusion manifold assembly via the skirt. In one embodiment, the microfluidic device is releasably engaged. In one embodiment, the microfluidic device is non-releasably engaged (e.g., a one-time connection), either via a locking mechanism or by using an adhesive (e.g., an adhesive layer to aid in the quality of the fluid seal). In one embodiment, the skirt has a guide mechanism for engaging with the microfluidic device. In one embodiment, the guide mechanism comprises a guide shaft, or a hole, groove, orifice, or other cavity configured to receive the guide shaft. In one embodiment, the guide mechanism comprises a guide track (either externally or internally). In one embodiment, the guide track is a side track. In one embodiment, the microfluidic device or chip is within a carrier, and the carrier is engaged with the perfusion manifold assembly through the side track of the skirt. In one embodiment, the microfluidic device has one or more features of the carrier to eliminate the need for additional substrates, such as carriers. In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels. In one embodiment, the assembly further comprises a cover or cover assembly for the fluid reservoir, the cover comprising a plurality of ports optionally associated with a filter. In some embodiments, the cover ports comprise through-holes, and the filters are positioned over corresponding holes in the gasket. In some embodiments, the cover comprises one or more channels passing through one or more of the ports (such that the ports are not simply through-holes).

In one embodiment, the present invention provides a fluid backplane comprising: a) a flow manifold assembly comprising: i) one or more fluid reservoirs; ii) a fluid backplane positioned below and in fluid communication with the fluid reservoirs, the fluid channels terminating in a fluid discharge port at a lower portion of the fluid backplane and a fluid resistor; and iii) a skirt or other protruding member having one or more side tracks; and b) a microfluidic device positioned on a carrier having one or more outer edges releasably engaging said one or more side tracks of said skirt, wherein said microfluidic device comprises i) microchannels in fluid communication with said perfusion manifold assembly via ii) one or more inlet ports on iii) a mating surface, wherein said one or more fluid inlet ports of said microfluidic device are positioned relative to said one or more fluid outlet ports of said perfusion manifold assembly, under conditions that allow fluid to flow from said fluid reservoir of said perfusion manifold assembly through said one or more fluid outlet ports to said one or more fluid inlet ports of said microfluidic device. In one embodiment, the carriers are releasably engaged. In one embodiment, the carriers are non-releasably engaged (e.g., a one-time connection), whether via a locking mechanism or using an adhesive (e.g., an adhesive layer to aid in the quality of a fluid seal). In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least portions of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels. In one embodiment, the assembly further comprises a cover for the fluid reservoir, the cover comprising a plurality of openings that associate with the channels. In one embodiment, the assembly further comprises a cover for the fluid reservoir, the cover comprising a plurality of ports that optionally associate with filters. In some embodiments, the cover ports comprise through-holes, and the filters are positioned over corresponding holes in the gasket. In some embodiments, the cover comprises one or more channels passing through one or more of the ports (such that the ports are not simply through-holes).

In one embodiment, the present invention provides a perfusion manifold assembly comprising: a) i) one or more fluid reservoirs, ii) a fluid backplane positioned below and in fluid communication with the fluid reservoirs, the fluid channels terminating in a skirt and a fluid resistor, iii) a skirt having one or more fluid discharge ports and one or more side tracks; and b) a microfluidic device positioned on a carrier having one or more outer edges releasably engaging the one or more side tracks of the skirt, wherein the microfluidic device comprises i) microchannels in fluid communication with the perfusion manifold assembly via ii) one or more inlet ports on the mating surface, wherein the one or more fluid inlet ports of the microfluidic device are positioned relative to the one or more fluid outlet ports of the skirt of the perfusion manifold assembly, under conditions that allow fluid to flow from the fluid reservoir of the perfusion manifold assembly through the one or more fluid outlet ports to the one or more fluid inlet ports of the microfluidic device. In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels. In a preferred embodiment, the microfluidic device comprises living cells that are perfused with fluid from the fluid reservoir. In one embodiment, the assembly further comprises a cover for the fluid reservoir, the cover comprising a plurality of ports optionally associated with a filter. In some embodiments, the cover ports comprise through-holes, and the filter is positioned over corresponding holes in the gasket. In some embodiments, the cover comprises one or more channels passing through one or more of the ports (such that the ports are not simply through-holes).

In a particularly preferred embodiment, the microfluidic device or chip comprises at least two different cell types that function together in a manner that mimics one or more functions of cells in an organ of the body (whether or not located on a carrier). In one embodiment, the microfluidic device comprises a membrane having upper and lower surfaces, wherein the upper surface comprises a first cell type and the lower surface comprises a second cell type. In one embodiment, the microfluidic device comprises an upper channel, a lower channel, and a membrane separating at least portions of the upper and lower channels. In one embodiment, the first cell type is an epithelial cell and the second cell type is an endothelial cell. In a preferred embodiment, the membrane is porous (e.g., porous to fluids, gases, cytokines, and other molecules, and in some embodiments porous to cells, thereby allowing cells to migrate through the membrane).

In one embodiment, the invention contemplates a method of seeding cells into a microfluidic chip (e.g., having ports associated with one or more microfluidic channels), comprising the steps of: a) providing a stand having a portion configured to receive i) a chip at least partially contained in a carrier, ii) cells, iii) a seeding guide, and iv) at least one seeding guide in a stable mounting position; b) engaging the seeding guide with the carrier to create an engaged seeding guide; c) mounting the engaged seeding guide on the stand, and d) seeding the cells onto the chip while the seeding guide is in the stable mounting position. In one embodiment, the seeding guide is configured to engage an edge of the carrier (e.g., utilizing a guide track). In one embodiment, the seeding guide comprises a side track (similar or identical to that on the skirt of an embodiment of a perfusion manifold assembly) for engaging an edge of the carrier. In one embodiment of the method, the plurality of seeding guides are mounted on a stand, allowing the plurality of chips to be seeded with cells. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, after the seeding, the microfluidic chip comprises cells on the membrane and/or within or on the channels. In one embodiment, the method further comprises, after the seeding of step d), the steps of e) separating the carrier from the seeding guide and f) engaging the perfusion manifold assembly with the carrier comprising the microfluidic chip comprising the cells.

In one embodiment, the invention contemplates a method of seeding cells onto a microfluidic chip (e.g., having ports that associate with one or more microfluidic channels), comprising the steps of: a) providing a stand having a portion configured to receive i) a chip at least partially contained in a seeding guide, ii) cells, and iii) at least one seeding guide in a stable mounting position; b) engaging the stand with the seeding guide; and c) seeding the cells onto the chip while the seeding guide is in the stable mounting position. In one embodiment of the method, a plurality of seeding guides are engaged with the stand, allowing a plurality of chips to be seeded with cells. In one embodiment of the method, there is no chip carrier. In another embodiment, the chip carrier acts as a seeding guide (without a separate seeding guide engaging the carrier).

In a preferred embodiment, when said one or more fluid inlet ports of said microfluidic device are positioned relative to said one or more fluid outlet ports of said perfusion manifold assembly, said carrier further comprises a locking mechanism for restricting movement of the carrier. The invention is not intended to be limited by the nature of the locking mechanism. In one embodiment, the locking mechanism is selected from the group consisting of a clip, a clamp, a stud, and a screw. In one embodiment, the locking mechanism engages by a friction fit. The locking mechanism can allow for a releasable engagement or a non-releasable engagement.

The present invention also contemplates a method of perfusing cells using a perfusion manifold assembly. In one embodiment, the present invention provides a method of perfusing cells, comprising: A) providing a microfluidic device positioned on a carrier comprising: a) i) one or more fluid reservoirs; ii) a fluid backplane positioned beneath and in fluid communication with said fluid reservoirs, said fluid channels terminating in an outlet port; and iii) a skirt or other protruding member comprising a guide mechanism; and b) i) living cells, and iii) microchannels on a mating surface, said microchannels in fluid communication with ii) one or more inlet ports, said microfluidic device being configured to engage the guide mechanism of said skirt; B) positioning said carrier so as to engage the guide mechanism of said skirt; And C) moving the carrier until the one or more fluid inlet ports of the microfluidic device are positioned relative to the one or more fluid outlet ports of the perfusion manifold assembly, under conditions whereby the microfluidic device is connected and fluid flows from the fluid reservoir of the perfusion manifold assembly through the one or more fluid outlet ports and into the one or more fluid inlet ports of the microfluidic device and the microchannels, thereby perfusing the cells. In one embodiment, the fluidic backplane comprises a fluidic resistor. In one embodiment, the guide mechanism comprises a guide shaft, or a hole, groove, orifice or other cavity configured to receive a guide shaft. In one embodiment, the guide mechanism comprises a guide track (either external or internal). In one embodiment, the guide track is a side track. In one embodiment, the carrier comprises one or more outer edges configured to engage the one or more side tracks of the skirt. In one embodiment, the movement of step C) comprises sliding the carrier along the side track until the inlet and outlet ports are positioned relative to one another. In one embodiment, the at least one inlet port on the mating surface of the microfluidic device comprises a droplet protruding above the mating surface, and the at least one outlet port on the perfusion manifold comprises a protruding droplet, such that the sliding of step C) causes a drop-to-drop connection. In one embodiment, the carriers are releasably engaged. In another embodiment, the carriers are non-releasably engaged (e.g., a one-time connection). In one embodiment, the assembly further comprises a cover or lid for the fluid reservoir, the cover comprising a plurality of ports optionally associated with a filter. In some embodiments, the cover ports comprise through-holes, and the filters are positioned over corresponding holes in the gasket. In some embodiments, the cover comprises at least one channel passing through at least one of the ports (such that the ports are not simply through-holes).

In one embodiment, the present invention provides a microfluidic device comprising the steps of: A) providing a perfusion manifold assembly comprising: i) one or more fluid reservoirs; ii) a fluid backplane in fluid communication with and positioned beneath the fluid reservoirs, the fluid channels terminating in a skirt and a fluid resistor; iii) a skirt having one or more fluid outlet ports and one or more side tracks; and b) a carrier having one or more outer edges configured to releasably engage with the one or more side tracks of the skirt, the carrier comprising i) living cells, and iii) microchannels in fluid communication with ii) one or more inlet ports on a mating surface; B) positioning the carrier such that the one or more outer edges engage with the one or more side tracks of the skirt; And C) sliding the carrier along the side track until the one or more fluid injection ports of the microfluidic device are positioned relative to the one or more fluid outlet ports of the skirt of the perfusion manifold assembly, under conditions whereby the microfluidic device is connected and fluid flows from the fluid reservoir of the perfusion manifold assembly through the one or more fluid outlet ports and into the one or more fluid injection ports of the microfluidic device and the microchannels, thereby perfusing the cells. In one embodiment, the one or more injection ports on the mating surface of the microfluidic device comprise a droplet protruding above the mating surface, and the one or more outlet ports on the skirt comprise a protruding droplet, such that when the one or more fluid injection ports of the microfluidic device are positioned relative to the one or more fluid outlet ports of the skirt of the perfusion manifold assembly, the sliding of step C) causes a drop-to-drop connection.

In one embodiment, the drop-to-drop connection does not allow air to enter one or more of the fluid injection ports. In one embodiment, the bonding surface proximate the drop is hydrophobic.

In one embodiment, the method further comprises the step of activating a locking mechanism to restrict movement of the carrier. In one embodiment, the method further comprises the step of installing the perfusion manifold assembly together with the connected microfluidic device within an incubator.

In one embodiment, the method (as described for any embodiment of the method of perfusion) further comprises installing the perfusion manifold assembly on, within, or in contact with the culture module together with the connected microfluidic device. In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a cover having a plurality of ports, and the culture module comprises a bonding surface having pressure points corresponding to the ports on the cover, such that installing the perfusion manifold assembly on or within the culture module together with the connected microfluidic device causes contact between the ports and the pressure points. In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a plurality of ports, and the culture module comprises a bonding surface having pressure points corresponding to the ports on the cover, such that after installing the perfusion manifold assembly on or within the culture module together with the connected microfluidic device, the pressure points of the bonding surface of the culture module contact the through-holes of the cover assembly. In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a cover having a plurality of through-hole ports that mate with corresponding holes in the strainer and the gasket, and the culture module comprises a bonding surface having pressure points corresponding to the through-hole ports on the cover, such that after the step of installing the perfusion manifold assembly on or into the culture module with the connected microfluidic device, the pressure points of the bonding surface of the culture module come into contact with the through-holes. In one embodiment, the fluid reservoir of the perfusion manifold assembly is covered with a cover assembly comprising a plurality of through-hole ports that mate with corresponding holes in the strainer and the gasket, and the culture module comprises a bonding surface having pressure points corresponding to the through-hole ports on the cover, such that after the step of installing the perfusion manifold assembly on or into the culture module with the connected microfluidic device, the pressure points of the bonding surface of the culture module come into contact with the through-holes of the cover assembly.

In one embodiment, the culture module comprises a volumetric controller. In one embodiment, the volumetric controller applies pressure to the fluid reservoir via the pressure point corresponding to the port on the cover. In one embodiment, the culture module comprises a pressure actuator. In one embodiment, the culture module comprises a pressure controller. In one embodiment, the pressure controller applies pressure to the fluid reservoir via the pressure point (e.g., on a pressure manifold) corresponding to the port (e.g., a through-hole port) on the cover. In one embodiment, the culture module comprises a plurality of perfusion manifold assemblies. In one embodiment, the culture module comprises an integrated valve. In one embodiment, the integrated valve is on the pressure manifold. In one embodiment, the valve comprises a Schrader valve.

The invention also contemplates a culture module as a device. In one embodiment, the device comprises an actuating assembly configured to move a plurality of microfluidic devices (e.g., a perfusion manifold assembly as described herein) relative to a pressure manifold comprising integrated valves. In one embodiment, the device is configured to move the microfluidic devices up relative to the non-moving pressure manifold. In one embodiment, the device comprises an actuating assembly configured to move one or more perfusion manifold assemblies into contact with the pressure manifold. In one embodiment, the device comprises an actuating assembly configured to move (either up or down) the pressure manifold into contact with the plurality of perfusion manifold assemblies. In some embodiments, the pressure manifold comprises integrated valves and an elastomeric membrane. In some embodiments, the elastic/flexible seal is disposed on the pod or cover rather than on the pressure manifold. In other embodiments, the invention is not considered limited to membranes because the membrane is described in only one particular way; In other embodiments, an o-ring, a gasket (thicker than the membrane), a flexible material, or vacuum grease is used instead. In one embodiment, the valve comprises a Schrader valve. In some embodiments, the pressure manifold is adapted to sense the presence of a coupled perfusion manifold assembly or microfluidic device, for example, to reduce pressure or fluid leakage in the absence of a coupled device. Importantly, in a preferred embodiment, the pressure manifold takes several pressure sources and distributes them to all of the perfusion manifold assemblies. In some embodiments, the pressure manifold is also designed to align directly with the perfusion manifold assembly (e.g., via alignment features on the pressure manifold mating surfaces). In one embodiment, the perfusion manifold assembly slides into the alignment features on the bottom of the pressure manifold, ensuring that the seal on the pressure manifold is always aligned with the ports on the perfusion manifold assembly. In some embodiments, when the pressure manifold is actuated, the pressure manifold has a set of springs that press down on the perfusion manifold assembly. These springs push the cover against the reservoir of the flow manifold assembly, creating a seal that maintains pressure (avoiding leaks) within the flow manifold assembly as pressure passes through the cover ports.

The present invention also contemplates a culture module and a perfusion disposable (PD) as a system. In one embodiment, the system comprises a device comprising an actuation assembly configured to move a plurality of microfluidic devices (e.g., a perfusion manifold assembly as described herein) relative to a pressure manifold comprising integrated valves. In one embodiment, the device comprises a) an actuation assembly configured to move a plurality of microfluidic devices (e.g., perfusion disposables) relative to a non-moving pressure manifold. In one embodiment, the system comprises a) a device comprising an actuation assembly configured to move a pressure manifold comprising integrated valves and seals (e.g., elastomeric membranes), and b) a plurality of microfluidic devices in contact with the seals (e.g., elastomeric membranes). In one embodiment, the microfluidic devices are perfusion disposables. In some embodiments, the elastomeric/flexible seals are disposed on a pod or cover rather than on the pressure manifold. In other embodiments, the invention is not considered limited to the membrane because the membrane is described in only one particular way; in other embodiments, an o-ring, a gasket (thicker than the membrane), a flexible material, or vacuum grease is used instead. In one embodiment, the valve comprises a Schrader valve. In one embodiment, the manifold uses a dual-stable engagement mechanism so that the actuator does not have to always provide engagement and sustained pressure against the cover. In a dual-stable mechanism, the actuator can engage the manifold and then turn off. This is useful in situations where the actuator generates excessive heat while powering it for long periods of time. In one embodiment, the perfusion disposable is interlocked with a microfluidic chip. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or in the membrane and/or the channels.

The present invention also contemplates a drop-to-drop connection arrangement for placing a microfluidic device in fluid communication with a fluid source or another microfluidic device, such as, but not limited to, placing a microfluidic device in fluid communication with a perfusion manifold assembly. In one embodiment, the present invention contemplates a fluidic device comprising a substrate having a first surface including one or more fluid ports, wherein the first surface is adapted to stably retain one or more liquid droplets comprising a first liquid in the one or more fluid ports. In one embodiment, the first surface comprises one or more regions surrounding the one or more fluid ports, the regions being adapted to resist wetting by the first liquid. In one embodiment, the regions are adapted to be hydrophobic. In one embodiment, the one or more regions comprise a first material selected to resist wetting by the first liquid. It is not intended that the present invention be limited to any particular first material. However, in one embodiment, the first material is selected from the group consisting of polytetrafluoroethylene (PTFE), perfluoroalkoxy alkane (PFA), fluorinated ethylenepropylene (FEP), polydimethylsiloxane (PDMS), nylon (some grades being hydrophilic and some being hydrophobic), polypropylene, polystyrene, and polyimide. In one embodiment, the substrate comprises the first material. In one embodiment, the first material is bonded, adhered, coated, or sputtered onto the first surface. In one embodiment, the first material comprises a hydrophobic gasket. In one embodiment, the one or more regions are adapted to resist wetting by the first liquid, such as by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents.

In one embodiment, the first surface comprises one or more regions surrounding one or more fluid ports, the regions being adapted to facilitate wetting by the first liquid. In one embodiment, the regions are adapted to be hydrophilic. In one embodiment, the one or more regions comprise a first material selected to facilitate wetting by the first liquid. Again, it is not intended that the invention be limited to any particular first material. However, in one embodiment, the first material is selected from the group consisting of polymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate (PC), polyether ether ketone (PEEK), polyethylene terephthalate (PET), polyfulphone, polystyrene, polyvinyl acetate (PVA), nylon, polyvinyl fluoride (PVF), polyvinylidene chloride (PVDC), polyvinyl chloride (PVC), and acrylonitrile-butadiene-styrene (ABS). In one embodiment, the substrate comprises the first material. In one embodiment, the first material is bonded, adhered, coated or sputtered onto the first surface. In one embodiment, the first material comprises a hydrophilic gasket. In one embodiment, the one or more regions are adapted to promote wetting by the first liquid by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents.

In one embodiment, the first surface comprises one or more raised portions surrounding one or more fluid ports. In one embodiment, the first surface comprises one or more depressed portions surrounding one or more fluid ports. In one embodiment, the first surface is adapted to stably retain one or more aqueous droplets. In one embodiment, the first surface is adapted to stably retain one or more non-aqueous droplets. In one embodiment, the first surface is adapted to stably retain one or more oil droplets.

The present invention also contemplates a system comprising a device for holding a droplet. In one embodiment, the system comprises: a) a first substrate comprising a first surface comprising a first set of one or more fluid ports, the first surface being adapted to stably hold one or more droplets comprising a first liquid in the first set of fluid ports; b) a second substrate comprising a second surface comprising a second set of one or more fluid ports; and c) a mechanism for fluidically contacting (connecting) the first set of fluid ports and the second set of fluid ports.

The present invention also contemplates a method of retaining a droplet that may be combined with establishing a fluid connection. In one embodiment, a method of establishing a fluid connection is contemplated, comprising the steps of: a) providing a first substrate comprising a first surface comprising a first set of fluid ports, the first surface being adapted to stably retain at least one droplet comprising a first liquid in the first set of fluid ports, b) providing a second substrate comprising a second surface comprising a second set of fluid ports, and c) contacting the first set of fluid ports and the second set of fluid ports (e.g., via controlled engagement). In a preferred embodiment, the contacting of step c) comprises aligning the first set of fluid ports and the second set of fluid ports and contacting the aligned sets of ports.

In one embodiment, the present invention contemplates a system and method for contacting a microfluidic device with a fluid source in a drop-to-drop connection. In one embodiment, the present invention contemplates a method comprising: a) providing a microfluidic device comprising i) a fluid source comprising a first protruding fluid droplet and in fluid communication with a first fluid port located on a first bonding surface; and ii) a microchannel comprising a second protruding fluid droplet and in fluid communication with a second fluid port located on a second bonding surface; and b) connecting the first protruding fluid droplet and the second fluid droplet together in drop-to-drop connection, such that fluid flows from the fluid source through the first fluid port to the second fluid port of the microfluidic device. In one embodiment, the present invention contemplates a method comprising: a) a fluid source adapted to support the first protruding fluid droplet and in fluid communication with a first fluid port located on the first bonding surface; b) a microfluidic device comprising a microchannel adapted to support a second protruding fluid droplet and in fluid communication with a second fluid port on a second bonding surface; and c) a mechanism wherein the first protruding fluid droplet and the second fluid droplet are connected drop-to-drop such that fluid can flow from the fluid source through the first fluid port to the second fluid port of the microfluidic device. In one embodiment, the first protruding fluid droplet projects downwardly from the first bonding surface and the second protruding fluid droplet projects upwardly from the second bonding surface. In one embodiment, the first protruding fluid droplet projects upwardly from the first bonding surface and the second protruding fluid droplet projects downwardly from the second bonding surface. In one embodiment, the mechanism raises the second bonding surface upwardly into contact with the first bonding surface. In another embodiment, the mechanism raises the first bonding surface upwardly into contact with the second bonding surface. In yet another embodiment, the mechanism lowers the second bonding surface into contact with the first bonding surface. In yet another embodiment, the mechanism lowers the first bonding surface into contact with the second bonding surface.

In one embodiment, the invention contemplates that the droplet is controlled by a surface treatment. In one embodiment of the system, the first bonding surface comprises a region surrounding the first fluid port, the region being adapted to resist wetting by the fluid. In one embodiment, the region is adapted to be hydrophobic. In one embodiment, the region comprises a first material selected to resist wetting by the fluid. It is not intended that the invention be limited to the properties of the first material. However, in one embodiment, the first material is selected from the group consisting of poly-tetrafluoroethylene (PTFE), perfluoroalkoxy alkane (PFA), fluorinated ethylenepropylene (FEP), polydimethylsiloxane (PDMS), nylon (some grades are hydrophobic), polypropylene, polystyrene, and polyimide. It is not intended that the invention be limited by the properties of the first material to which it adheres to the surface. However, in one embodiment, the first material is bonded, adhered, coated, or sputtered onto the first bonding surface. The present invention also contemplates adding a feature having an inherently hydrophobic surface, or a surface capable of being rendered hydrophobic. In one embodiment, the first material comprises a hydrophobic gasket. The present invention is not intended to be limited to the particular treatment method utilized to modify the surface or a region of the surface. However, in one embodiment, the region of the first bonding surface is adapted to resist wetting, such as by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents.

While embodiments in which a surface or region of a surface is adapted to resist wetting have been discussed above, the present invention contemplates embodiments in which the first bonding surface comprises a region surrounding the first fluid port, the region being adapted to facilitate wetting by the fluid. In one embodiment, the region is adapted to be hydrophilic. In one embodiment, the region comprises a first material selected to facilitate wetting by the first liquid. It is not intended that the present invention be limited to particular first materials that facilitate wetting. However, in one embodiment, the first material is selected from the group consisting of polymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate (PC), polyether ether ketone (PEEK), polyethylene terephthalate (PET), polyfulphone, polystyrene, polyvinyl acetate (PVA), nylon (some grades are hydrophilic), polyvinyl fluoride (PVF), polyvinylidene chloride (PVDC), polyvinyl chloride (PVC), and acrylonitrile-butadiene-styrene (ABS). Furthermore, the present invention is not intended to be limited by the technique by which the first material is attached to the surface. However, in one embodiment, the first material is bonded, adhered, coated, or sputtered onto the first bonding surface. The present invention also contemplates introducing structures or features having an inherently hydrophilic surface, or a surface that can be made hydrophilic. For example, in one embodiment, the first material comprises a hydrophilic gasket. Furthermore, the present invention is not intended to be limited to treatment modalities that promote wetting. For example, in one embodiment, said region of said first bonding surface is adapted to promote wetting by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents.

The present invention also contemplates structural and geometric features that can be molded or formed as part of the surface, attached to, deposited on, printed on, or bonded to the source, or machined, etched, or abraded into the surface. For example, in one embodiment, the first mating surface comprises one or more raised portions surrounding the first fluid port. In another embodiment, the first mating surface comprises one or more depressed portions surrounding the first fluid port.

The present invention is also not limited to drop-to-drop connections with only aqueous fluids. In one embodiment, the first bonding surface is adapted to stably hold an aqueous protruding fluid droplet, while in another embodiment, the first bonding surface is adapted to stably hold a non-aqueous protruding fluid droplet, such as, but not limited to, an oily protruding droplet.

The present invention also contemplates a method of fusing droplets using a drop-to-drop approach. In one embodiment, the present invention contemplates a method of fusing droplets, comprising: a) providing a microfluidic device or chip comprising: i) a fluid source comprising a first protruding fluid droplet and in fluid communication with a first fluid port located on a first bonding surface; and ii) a microchannel comprising a second protruding fluid droplet and in fluid communication with a second fluid port located on a second bonding surface; and b) connecting the first protruding fluid droplet and the second fluid droplet together drop-to-drop, such that the first and second fluid drops fuse, thereby allowing fluid to flow from the fluid source through the first fluid port to the second fluid port of the microfluidic device. In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on or within the membrane and/or the channels. The invention is not intended to be limited to a particular orientation or two bonding surfaces. In one embodiment, the first protruding fluid droplet protrudes downwardly from the first bonding surface, and the second protruding fluid droplet protrudes upwardly from the second bonding surface. In another embodiment, the first protruding fluid droplet protrudes upwardly from the first bonding surface, and the second protruding fluid droplet protrudes downwardly from the second bonding surface. The invention is not intended to be limited to a method of bringing the droplets together. In one embodiment, step b) comprises elevating the second bonding surface upwardly into contact with the first bonding surface. In another embodiment, step b) comprises elevating the first bonding surface upwardly into contact with the second bonding surface. In yet another embodiment, step b) comprises lowering the second bonding surface downwardly into contact with the first bonding surface. In yet another embodiment, step b) comprises lowering the first bonding surface downwardly into contact with the second bonding surface. In a preferred embodiment, the drop-to-drop connection does not permit air to enter the fluid injection port.

The present invention contemplates a surface treatment to promote wetting. In one embodiment, the first bonding surface comprises a region surrounding the first fluid port, the region being adapted to promote wetting by the fluid. In one embodiment, the region is adapted to be hydrophilic. In one embodiment, the region comprises a first material selected to promote wetting by the fluid. While it is not intended to limit the invention to any particular first material, in one embodiment, the first material is selected from the group consisting of polymethylmethacrylate (PMMA), polyvinyl alcohol (PVOH), polycarbonate (PC), polyether ether ketone (PEEK), polyethylene terephthalate (PET), polyfulphone, polystyrene, polyvinyl acetate (PVA), nylon, polyvinyl fluoride (PVF), polyvinylidene chloride (PVDC), polyvinyl chloride (PVC), and acrylonitrile-butadiene-styrene (ABS). While not intended to limit the present invention to any particular attachment approach, in one embodiment, the first material is bonded, adhered, coated or sputtered onto the first bonding surface.

In some embodiments, the present invention contemplates adding features or structures to a surface, including structures having an inherently hydrophilic surface (or a surface capable of becoming hydrophilic). In one embodiment, the first material comprises a hydrophilic gasket.

The present invention is not intended to be limited to any particular surface treatment technique. However, in one embodiment, said region of said first bonding surface is adapted to promote wetting by plasma treatment, ion treatment, vapor deposition, liquid phase deposition, adsorption, absorption, or chemical reaction with one or more agents.

Additional structures may be molded or otherwise formed within or on the surface. For example, in one embodiment, the first mating surface comprises one or more raised portions surrounding the first fluid port. In another embodiment, the first mating surface comprises one or more depressed portions surrounding the first fluid port.

As noted above, the fluid need not be an aqueous fluid. In one embodiment, the present invention contemplates that the first bonding surface is adapted to stably hold an aqueous protruding fluid droplet, while in another embodiment, the first bonding surface is adapted to stably hold a non-aqueous protruding fluid droplet, such as, but not limited to, an oily protruding fluid droplet.

The present invention also contemplates a system for connecting ports together. In one embodiment, the system comprises a) a first substrate comprising a first fluid port, b) a second substrate comprising a second fluid port, c) a guide mechanism adapted to align the first port and the second port, and (optionally) d) a retaining mechanism adapted to maintain contact between the first substrate and the second substrate. While the present invention is not intended to be limited to any particular guide mechanism, in one embodiment the guide mechanism is a guide track positioned on the first substrate, the guide track configured to engage a portion of the second substrate. The present invention contemplates embodiments in which the retaining mechanism is on either the first or second substrate, but in one embodiment the retaining mechanism is a clip positioned on the second substrate, the clip configured to engage the first substrate.

In another embodiment, the present invention contemplates a system comprising a) a first substrate comprising a first set of one or more fluid ports, b) a second substrate comprising a second set of one or more fluid ports, c) a guide mechanism adapted to align the first set of ports and the second set of ports, and d) a retaining mechanism adapted to maintain contact between the first substrate and the second substrate. Again, a variety of guide mechanisms are contemplated (and are discussed herein). In one embodiment, the guide mechanism comprises a guide shaft, or a hole, groove, orifice or other cavity configured to receive the guide shaft. However, in one embodiment, the guide mechanism is a guide track disposed on the first substrate, the guide track being configured to engage a portion of the second substrate. Again, a variety of retaining mechanisms are contemplated (and are discussed herein). However, in one embodiment, the retaining mechanism is a clip disposed on the second substrate, the clip being configured to engage the first substrate.

The present invention also contemplates a method of connecting ports in a manner that establishes a fluid connection. In one embodiment, the present invention contemplates a method of establishing a fluid connection, comprising the steps of: a) providing a first substrate comprising a first fluid port, a second substrate comprising a second fluid port, and a guide mechanism adapted to guide the second substrate, b) engaging the second substrate with the guide mechanism, c) aligning the first and second sets of fluid ports with the aid of the guide mechanism, and d) contacting the first and second fluid ports, thereby establishing the fluid connection. While various guide mechanisms are contemplated, in one embodiment the guide mechanism comprises a guide track positioned on the first substrate, the guide track configured to engage a portion of the second substrate. In one embodiment of this method of establishing a fluid connection, the second substrate comprises a microfluidic device comprising a bonding surface, the second fluid port comprising a droplet positioned on the bonding surface and projecting above the bonding surface. In a further embodiment, the first substrate comprises a bonding surface, and the first fluid port is positioned on the bonding surface and comprises a protruding droplet. In this embodiment still further, the contacting of step d) causes a drop-to-drop connection when the first and second fluid ports establish a fluid connection. Preferably, the drop-to-drop connection does not allow air to enter one or more of the fluid injection ports. While the invention is not limited to the manner of alignment, in one embodiment the alignment of step c) comprises sliding the second substrate by a guide track. While various designs and configurations for the guide track are contemplated, in one embodiment the guide track comprises first and second compartments, the first compartment being formed to support the alignment of step c) and the second compartment being formed to support the contacting of step d).

The present invention contemplates embodiments in which the retaining mechanism is on the first substrate, but in one embodiment, the second substrate comprises a retaining mechanism adapted to maintain contact between the first substrate and the second substrate. In some embodiments, the retaining mechanism is automatically engaged when the first and second substrates are brought into contact to establish a fluid connection. However, in one embodiment, the present invention comprises an activation step of e) activating the retaining mechanism.

While a two-substrate system is described above, the present invention also contemplates a three-substrate system. In one embodiment, the system comprises: a) a first substrate comprising a first fluid port, b) a second substrate comprising a second fluid port, c) a third substrate configured to support the second substrate; d) a guide mechanism adapted to align the first port relative to the second port, and e) a holding mechanism means adapted to maintain contact between the first substrate and the second substrate.

As previously mentioned, various guide mechanisms are contemplated (and discussed herein). In one embodiment, the guide mechanism comprises a guide shaft, or a hole, groove, orifice, or other cavity configured to receive the guide shaft. There may be one or more guide shafts or other protrusions on one substrate, and one or more holes, grooves, orifices, or other cavities on the other substrate may be configured to receive the one or more guide shafts or other protrusions. In one embodiment, the guide mechanism comprises a guide track. The guide track(s) may have any orientation (e.g., coming from above rather than from a side). While the present invention contemplates that the guide mechanism may be attached to the first, second, or third substrate, in one embodiment, the guide track is located on the first substrate. While the present invention contemplates embodiments in which the second or third substrate has features or structures configured to engage the guide mechanism, in one embodiment, the present invention contemplates that the third substrate includes an edge configured to engage the guide track. In one embodiment, the second substrate comprises an edge configured to engage with the guide track. While the present invention contemplates embodiments in which the retaining mechanism is positioned on the first or second substrate, in one embodiment, the retaining mechanism is positioned on the third substrate. As previously noted, a variety of retaining mechanisms are contemplated. In one embodiment, the retaining mechanism comprises a clip configured to engage with the first substrate. In another embodiment, the retaining mechanism comprises a clamp configured to engage with the first substrate under conditions such that contact between the first and second substrates is maintained. In yet another embodiment, the retaining mechanism comprises a stud configured to engage with a hole in the first substrate. In yet another embodiment, the retaining mechanism is frictionally engaged with a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of an adhesive (e.g., a laminate), a heat stake, and a screw.

While the present invention contemplates a system in which the components of the system are as described (see above), the present invention also contemplates an assembly in which the components are arranged, attached, or connected in a particular manner. In one embodiment, the present invention contemplates an assembly comprising: a) a first substrate comprising a first fluid port and a guide mechanism and positioned in contact with a second substrate; b) a second substrate comprising a second fluid port and supported by a carrier; c) a carrier comprising a portion of said first substrate that engages said guide mechanism, wherein said first and second ports are aligned to permit fluid communication. While the present invention contemplates embodiments in which the retaining mechanism is positioned on said first or second substrate, in one embodiment, the carrier further comprises a retaining mechanism for maintaining said contact between said first and second substrates. While various guide mechanisms are contemplated (as described herein), in one embodiment, the guide mechanism comprises a guide track. The present invention is not limited to a single guide track, and two or more guide tracks may be used. For example, in one embodiment, the guide track is positioned on one or more sides of said first substrate. In one embodiment, the carrier portion that engages the first substrate includes one or more edges configured to engage the guide tracks.

While various retaining mechanisms are contemplated (as described herein), in one embodiment of the assembly, the retaining mechanism comprises a clip configured to engage with the first substrate. In another embodiment, the retaining mechanism comprises a clamp configured to engage with the first substrate. In still another embodiment, the retaining mechanism comprises a stud configured to engage with a hole in the first substrate. In a particular embodiment, the retaining mechanism frictionally engages a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of an adhesive (such as, but not limited to, a laminate), a heat stake, and a screw.

The present invention also contemplates a method of establishing a fluid connection by assembling fluid ports comprising three substrates. In one embodiment, the present invention contemplates a method of establishing a fluid connection comprising the steps of: a) providing a first substrate comprising a first fluid port, a second substrate comprising a second fluid port, a third substrate configured to support the second substrate, and a guide mechanism; b) aligning the first and second ports with the guide mechanism; and c) contacting the first port and the second port under conditions such that a fluid connection is established between the first and second substrates. In one embodiment of the three substrate method, the second substrate comprises a microfluidic device comprising a bonding surface, wherein the second fluid port comprises a droplet positioned on the bonding surface and projecting above the bonding surface. In this embodiment further, the first substrate comprises a bonding surface, and the first fluid port comprises a droplet positioned on the bonding surface and projecting. In this embodiment still further, the contacting of step c) causes a point-to-point connection when the first and second fluid ports establish a fluid connection. Preferably, the point-to-point connection does not allow air to enter one or more of the fluid injection ports.

Again, various guide mechanisms are contemplated and described herein. In one embodiment, the guide mechanism comprises a guide track. The present invention contemplates that the guide track is positioned on the first, second or third substrate, although in a preferred embodiment, the guide track is positioned on the first substrate. In one embodiment, the third substrate comprises an edge configured to engage the guide track. While the present invention is not intended to be limited to a particular technique for alignment, in one embodiment, the present invention contemplates that the alignment of step b) comprises sliding the third substrate by the guide track. In one embodiment, the guide track comprises first and second sections, the first section being configured to support the alignment of step b) and the second section being configured to support the contact of step c). In one embodiment, the first section is linear and the second section is curved. In yet another embodiment, the guide mechanism comprises a mechanism that causes the third substrate to rotate or pivot during step d). For example, in one embodiment, the guide mechanism comprises a hinge, joint, or pivot point.

While the present invention contemplates embodiments in which the retaining mechanism is positioned on the first or second substrate, in one embodiment, the present invention further comprises a retaining mechanism for maintaining alignment of the first and second ports with the third substrate. Again, various retaining mechanisms are contemplated. In one embodiment, the retaining mechanism comprises a clip configured to engage with the first substrate. In one embodiment, the retaining mechanism comprises a clamp configured to engage with the first substrate under conditions such that contact between the first and second substrates is maintained. In yet another embodiment, the retaining mechanism comprises a stud configured to engage with a hole on the first substrate. In yet another embodiment, the retaining mechanism is frictionally engaged with a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of an adhesive (such as, but not limited to, a laminate), a heat stake, and a screw. The present invention also contemplates embodiments in which the third substrate is a carrier for the second substrate.

In one embodiment, the present invention contemplates a method of establishing a fluid connection, comprising: a) providing a first substrate including a guide mechanism and a first fluid port on a first bonding surface, a second substrate including a second fluid port on a second bonding surface and a lower surface, and a carrier including a retaining mechanism and at least one edge for engaging the guide mechanism and contacting the lower surface of the second substrate; b) engaging the guide mechanism of the first substrate with the at least one edge of the carrier; c) aligning the first and second ports with the guide mechanism; and d) contacting the first bonding surface and the second bonding surface under conditions such that the first port contacts the second port such that a fluid connection is established between the first and second substrates. In one embodiment of the method, the second fluid port comprises a droplet protruding above the bonding surface of the second substrate. In one embodiment, the first fluid port comprises a protruding droplet. In one embodiment, the contacting of step d) causes a drop-to-drop connection when the first and second fluid ports establish a fluid connection. Preferably, the drop-to-drop connection does not allow air to enter one or more of the fluid injection ports. While various guide mechanisms are contemplated, in one embodiment, the guide mechanism comprises a guide track. The invention is not limited to embodiments having only one guide track, and two or more guide tracks may be used. In one embodiment, the guide track is positioned on one or more sides of the first substrate. In a preferred embodiment, the carrier comprises one or more edges configured to engage the guide track. While various alignment approaches are contemplated, in one embodiment, the alignment of step c) comprises sliding the carrier by means of the guide track. While various designs and configurations for the guide track are contemplated, in one embodiment, the guide track comprises first and second compartments, the first compartment being formed to support the alignment of step c) and the second compartment being formed to support the contacting of step d). In one embodiment, the first section is linear and the second section is curved. In yet another embodiment, the guide mechanism comprises a mechanism that causes the carrier to rotate or pivot during step d). In this embodiment, the guide mechanism can comprise a hinge, a joint, a socket or other pivot point.

In some embodiments, the retaining mechanism automatically engages when or after contact is made in step d). However, in one embodiment, the invention further comprises the step of e) activating the retaining mechanism under conditions such that the alignment of the first and second ports is maintained. Again, various retaining mechanisms are contemplated. In one embodiment, the retaining mechanism comprises a clip configured to engage the first substrate. In one embodiment, the retaining mechanism comprises a clamp configured to engage the first substrate under conditions such that contact between the first and second substrates is maintained. In one embodiment, the retaining mechanism comprises a stud configured to engage a hole in the first substrate. In one embodiment, the retaining mechanism frictionally engages a portion of the first substrate. In one embodiment, the retaining mechanism is selected from the group consisting of an adhesive (such as, but not limited to, a laminate), a heat stake, and a screw.

The invention also contemplates a device for perfusing cells, such as a device for applying pressure to a fluid reservoir to produce a flow of fluid (e.g., culture medium). In one embodiment, the invention contemplates a device comprising an actuating assembly configured to move a pressure manifold comprising an integrated valve. In one embodiment, the device further comprises an elastomeric membrane. In one embodiment, the valve comprises a Schrader valve. In one embodiment, the pressure manifold comprises a mating surface having a pressure point. In one embodiment, the device further comprises a pressure controller. In one embodiment, the pressure controller is configured to apply pressure across the pressure point. In one embodiment, the actuating assembly comprises a pneumatic cylinder operably connected to the pressure manifold. In one embodiment, the microfluidic device or chip further comprises an alignment feature configured to align the mating surface with the microfluidic device or chip when the microfluidic device or chip is engaged with the mating surface. In one embodiment, the device is a culture module for perfusing cells. In one embodiment, the microfluidic chip is interlocked with a perfusion manifold assembly (the alignment features being configured to align the perfusion manifold assembly). In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least portions of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels.

The present invention also contemplates a system wherein a device for transmitting pressure is connected to a plurality of microfluidic devices, more preferably, the plurality of microfluidic devices (e.g., various embodiments of perfusion disposables discussed herein) are connected simultaneously (although they may be connected individually or sequentially, if desired). In one embodiment, the present invention contemplates a system comprising a) a device comprising an actuation assembly configured to move a pressure manifold comprising an integrated valve, wherein the pressure manifold is in contact with b) a plurality of microfluidic devices (e.g., various embodiments of perfusion disposables discussed herein). In one embodiment, the pressure manifold further comprises an elastomeric membrane, wherein the elastomeric membrane is in contact with the microfluidic device. In one embodiment, the microfluidic device is a perfusion disposable. In one embodiment, the valve comprises a Schrader valve. In one embodiment, each of the microfluidic devices is covered by a cover assembly comprising a cover having a plurality of ports, the pressure manifold comprising a mating surface having pressure points corresponding to the ports on the cover, the pressure points of the mating surface of the pressure manifold contacting the ports of the cover assembly. In one embodiment, the ports comprise through-hole ports that mate with corresponding holes in the strainer and the gasket. In one embodiment, the device further comprises a pressure controller. In one embodiment, the pressure controller is configured to apply pressure through the pressure points. In one embodiment, the actuation assembly comprises a pneumatic cylinder operably connected to the pressure manifold. In one embodiment, the mating surface of the pressure manifold further comprises an alignment feature configured to align the microfluidic device when the microfluidic device is engaged with the mating surface. In a preferred embodiment, the device is a culture module for perfusing cells. In one embodiment of such a culture module, the culture module is configured to receive one or more trays, each tray comprising a plurality of microfluidic devices. In one embodiment, the culture module further comprises a user interface for controlling the culture module. In one embodiment, each tray comprises a plurality of perfusion manifold assemblies. In one embodiment, the microfluidic chip is interdigitated with each of the perfusion manifold assemblies (the alignment features of the pressure manifold junction surfaces are configured to align each of the perfusion manifold assemblies). In one embodiment, the microfluidic chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device comprises cells on the membrane and/or within or on the channels.

The present invention also contemplates methods of perfusing cells (e.g., cells in microchannels of a microfluidic device, such as the various embodiments of the perfusion disposables discussed herein, wherein the cells are first seeded into the microfluidic device with or without the use of a seeding guide of the type described herein) using a culture module. In one embodiment, the present invention provides a method comprising: A) providing a culture module comprising: a) i) an actuation assembly configured to move a plurality of microfluidic devices relative to a pressure manifold, ii) a pressure manifold comprising a bonding surface having pressure points; and b) a plurality of microfluidic devices, each of the microfluidic devices comprising i) one or more microchannels comprising living cells, ii) one or more reservoirs comprising culture medium, and iii) a cover assembly over the one or more reservoirs, the cover having ports corresponding to the pressure points on the pressure manifold bonding surface; B) mounting the plurality of microfluidic devices on or within the culture module; And C) simultaneously (or sequentially) contacting the ports on the covers of each of the plurality of microfluidic devices with the mating surfaces of the pressure manifold, such that culture medium flows from the reservoir into the microchannels of the microfluidic device, thereby perfusing the cells. In one embodiment, the plurality of microfluidic devices are positioned on one or more trays prior to step B), and the placing of step B) comprises simultaneously moving at least a subset of the plurality of microfluidic devices into the culture module. In one embodiment, the simultaneous contacting of step C) comprises moving the plurality of microfluidic devices upwardly relative to the mating surfaces of the pressure manifold via an actuation assembly. In another embodiment, the invention contemplates a method of perfusing cells, comprising: A) a culture module comprising: a) i) an actuation assembly configured to move the pressure manifold, ii) a pressure manifold comprising a mating surface having a pressure point; And b) providing a plurality of microfluidic devices, each of said microfluidic devices comprising i) one or more microchannels containing living (viable) cells, ii) one or more reservoirs containing culture medium, and iii) a cover having a port corresponding to a pressure point on a pressure manifold junction surface, wherein the cover assembly is over said one or more reservoirs; B) mounting said plurality of microfluidic devices on or within said culture module; and C) simultaneously (or sequentially) contacting said ports on the covers of each of said plurality of microfluidic devices with the junction surface of said pressure manifold, thereby causing said ports to contact said pressure points under conditions such that culture medium flows from said reservoirs into said microchannels of said microfluidic device, thereby perfusing said cells. In said embodiments, the plurality of microfluidic devices are simultaneously connected. They can then be simultaneously disconnected or decoupled from the pressure manifold. In one embodiment, the plurality of microfluidic devices are positioned on one or more trays (or nests) prior to step B), and the installation of step B) comprises simultaneously moving at least a subset (at least three) of the plurality of microfluidic devices into the culture module. In one embodiment, the simultaneous contacting of step C) is accomplished by downwardly moving the mating surface of the pressure manifold via the actuation assembly onto the cover assembly of the plurality of microfluidic devices. In one embodiment of the perfusion method, the microfluidic device comprises a microfluidic chip (such as, but not limited to, the microfluidic chip illustrated in FIG. 3A having one or more microchannels and ports) interlocked with a perfusion manifold assembly, the assembly comprising i) a cover or lid configured to act as an upper portion of one or more fluid reservoirs, ii) one or more fluid reservoirs, iii) a fluidic backplane in fluidic communication with and beneath the fluid reservoir(s), and iv) a protruding member or skirt interlocked (directly) with the microfluidic chip or (indirectly through) with a carrier containing the microfluidic chip. The perfusion is preferably performed at a rate that provides (or maintains) a viability of greater than 80%, and more preferably greater than 90%, and most preferably greater than 95% of the cells contained within the microfluidic chip. In one embodiment, the assembly further comprises a capping layer underlying the fluidic reservoir(s). In one embodiment, the fluidic backplane comprises a resistor. In a preferred embodiment, the microfluidic chip environment remains sterile during the perfusion.

The present invention also contemplates controlling the pressure while perfusing cells (e.g., cells in microchannels of a microfluidic device, such as the various embodiments of perfusion disposables discussed herein, wherein the cells are first seeded into the microfluidic device with or without the use of a seeding guide of the type described herein), such as in one embodiment controlling the pressure such that the pressure is reliably maintained at 1 pKa (+ or - 0.5 pKa, and more preferably + or - 0.15 pKa). In one embodiment, the invention contemplates a method of controlling pressure while perfusing cells, comprising the steps of A) providing a) a plurality of microfluidic devices, b) one or more pressure actuators, each of the microfluidic devices comprising i) one or more microchannels comprising living cells, ii) one or more reservoirs comprising culture medium, B) coupling the pressure actuator to at least one of the reservoirs, wherein the coupling is adapted to control perfusion of at least a portion of the living cells at an actuated pressure, C) turning on the one or more pressure actuators between two or more pressure setpoints, thereby controlling pressure while perfusing the cells. In another embodiment, the invention contemplates a method of controlling pressure while perfusing cells, comprising: A) a culture module comprising: a) i) an actuation assembly configured to move a pressure manifold, ii) a pressure manifold comprising a bonding surface having a pressure point, and iii) one or more pressure controllers for providing a pressure to the pressure point; And b) providing a plurality of microfluidic devices, each of the microfluidic devices comprising i) one or more microchannels containing living cells, ii) one or more reservoirs containing culture medium, and iii) a cover having a port corresponding to a pressure point on a pressure manifold junction surface, the cover assembly over the one or more reservoirs; B) mounting the plurality of microfluidic devices on or within the culture module; C) simultaneously contacting the ports on the covers of each of the plurality of microfluidic devices with the junction surface of the pressure manifold, thereby causing the ports to contact the pressure points under conditions such that culture medium flows from the reservoirs into the microchannels of the microfluidic device, thereby perfusing the cells; and D) controlling the pressure while perfusing the cells, the method comprising: turning the one or more pressure controllers off for a predetermined period of time and on for a predetermined period of time (or switching them off and on) (or switching them off and on between two or more setpoints). In one embodiment, the switching is between the setpoints 1 kPa and 0.5 kPa, with good conversion within that range. In one embodiment, the switching has three levels in some advanced methods: 2 kPa, 1 kPa and 0 kPa. In one embodiment, the pressure controller is turned on and off (or between the setpoints) in a switching pattern (e.g., turning them off and on, or repeatedly turning them on and off at defined intervals between the setpoints). In a preferred embodiment, the switching pattern is selected such that the average value of the liquid working pressure in the one or more reservoirs corresponds to the desired value. For cells, the desired value is typically low. For example, in one embodiment, the switching pattern is selected such that the average gas pressure is maintained below 1 kPa. In one embodiment of the method of perfusion and pressure control, the microfluidic device comprises a microfluidic chip (such as, but not limited to, the microfluidic chip illustrated in FIG. 3A having one or more microchannels and ports) mated to a perfusion manifold assembly, the assembly comprising i) a cover or lid configured to act as an upper portion of one or more fluid reservoirs, ii) one or more fluid reservoirs, iii) a fluidic backplane in fluid communication with and beneath the fluid reservoir(s), and iv) a protruding member or skirt that either (directly) engages with the microfluidic chip or (indirectly through) engages with a carrier containing the microfluidic chip. The perfusion is preferably performed at a rate that provides a viability of greater than 80%, and more preferably greater than 90%, and most preferably greater than 95% of the cells contained within the microfluidic chip. In one embodiment, the assembly further comprises a capping layer beneath the fluid reservoir(s). In one embodiment, the fluidic backplane comprises a resistor. In one embodiment, the port on the cover or lid is associated with a filter. In one embodiment, the filter is a 0.2 micrometer, 0.4 micrometer or 25 micrometer filter. In a preferred embodiment, the microfluidic chip environment remains sterile during said perfusion. In one embodiment, cycling the pressure regulator on and off brings the average pressure value closer to the desired value, but by introducing a resistive filter into the inlet portion in the lid of the perfusion manifold assembly, the maximum and minimum values seen by the microfluidic device or chip are much closer to the desired values.

A pressure cover is contemplated as a device within or otherwise associated with a microfluidic device that allows for pressurization of one or more fluid sources (e.g., reservoirs). In one embodiment, the invention contemplates a pressure cover comprising a plurality of ports configured to mate with a pressure manifold. In one embodiment, the ports are associated with a filter. In one embodiment, the cover is associated with a gasket. In one embodiment, the pressure cover is movable or removably attached to the microfluidic device to allow improved access to the member (e.g., reservoir) therein. In one embodiment, the invention provides a method comprising: a) providing a pressure cover comprising a plurality of ports configured to mate with a pressure manifold, a microfluidic device comprising a fluid source, and a pressure manifold; b) positioning the pressure cover over the fluid source to create a positioned pressure cover; and c) engaging the positioned pressure cover with the pressure manifold under conditions whereby pressure is applied through the port such that fluid from the fluid source moves into or through the microfluidic device. In one embodiment, the method further comprises the step of d) detaching the positioned pressure cover from the pressure manifold. The pressure cover can then be (optionally) removed, and the microfluidic device can be used without the cover.

The present invention also contemplates a system comprising a) a device for interfacing with a microfluidic device, b) a microfluidic device comprising or in fluid communication with i) one or more fluid reservoirs, and ii) a pressure sheath comprising one or more device-facing ports and one or more reservoir-facing ports, wherein the pressure sheath is adapted to transfer a pressure between at least one of the device-facing ports and at least one of the reservoir-facing ports. In one embodiment, the device comprises a pressure manifold (which may or may not be movable). In one embodiment, the one or more fluid reservoirs are disposed in a cartridge, the cartridge being in fluid communication with the microfluidic device. In one embodiment, the one or more fluid reservoirs are disposed in the microfluidic device.

The present invention also contemplates a pressure cover comprising at least one equipment-facing port and at least one reservoir-facing port as a device, the pressure cover being adapted to transfer a pressure between at least one of the equipment-facing ports and at least one of the reservoir-facing ports, the pressure cover being adapted to form a pressure connection with at least one fluid reservoir.

The present invention also contemplates a pressure cover comprising one or more channels as a device, each channel including a device-connection end and a reservoir-connection end, the channels being configured to transmit pressure between the device and a fluid reservoir.

FIG. 1A is an exploded view of an embodiment of a perfusion manifold assembly, showing a cover (or cover assembly) of a reservoir (the reservoir body may be made of, for example, acrylic), a reservoir positioned over a backplane, the backplane in fluid communication with the reservoir, a skirt having side tracks for engaging a representative microfluidic device or "chip" (which may be made of, for example, a plastic, such as PDMS) having one or more inlet, outlet, and (optionally) vacuum ports, and one or more microchannels, a chip carrier (which may be made of, for example, a thermoplastic polymer, such as acrylonitrile butadiene styrene (ABS)), the chip depicted next to (but not within) an embodiment of the chip, a carrier configured to support and retain the chip, for example having dimensions such that the chip fits within the cavity. FIG. 1B is an identical embodiment of a perfusion manifold assembly having chips within a chip carrier that is fully connected to the reservoir and the cover, and the skirt of the perfusion manifold assembly, in fluid communication with the reservoir. In one embodiment, each chip has two inlets, two outlets, and (optionally) two connections for vacuum stretch. In one embodiment, when the chips are in fluid communication, they are connected in one operation rather than one at a time. FIG. 1C is an exploded view (before the components are assembled) of one embodiment of a perfusion manifold assembly including a reservoir that is fluidly sealed by a capping layer and positioned over a fluid backplane (including a fluid resistor) positioned over the skirt, the individual pieces being dimensioned to fit together. In one embodiment, the skirt has a structure (e.g., made of a polymer) that borders or defines two open spaces, one of the spaces being configured to receive a carrier having a chip therein. In one embodiment, the skirt has a structure that completely surrounds two "arms" extending outwardly to define one open space and a second open space for receiving a carrier. In one embodiment, the two arms have side tracks that slidably engage the carrier edges.
FIG. 2A is an exploded view of one embodiment of a pressure cover or cover assembly including a pressure cover. In the depicted embodiment, the pressure cover includes a plurality of ports (e.g., through-hole ports) that mate with corresponding apertures in the strainer and gasket. The depicted design of the apertures in the gasket is intended to assist the gasket in holding the depicted strainer in place. In alternative embodiments, the gasket openings may utilize a different configuration than the apertures in the cover. For example, the gasket may be shaped to conform to the contour of one or more reservoirs intended to form a fluid or pressure seal. In some embodiments, a plurality of gaskets may be utilized. In some embodiments, the strainer and/or gasket may be secured, clamped, or captured by the absence of the cover and/or additional substrate using adhesives, heat stakes, bonding (ultrasonic, solvent-assisted, laser welding). While the depicted pressure cover includes through-hole ports, an alternative embodiment includes one or more channels passing from at least one upper surface port to one or more lower surface ports, wherein the lower surface ports need not be directly beneath the upper surface ports. FIG. 2b illustrates the same embodiment of the cover assembly depicted in FIG. 2a, with a strainer and a gasket within (and beneath) the cover. FIG. 2c-1 is a cross-sectional view of one embodiment of the cover assembly, illustrating a raised portion or sealing tooth surrounding both of the through-hole ports in the cover. FIG. 2c-2 is an enlarged view of a portion (circled) of FIG. 2c-1. In the depicted embodiment, the cross-sectional shape of the sealing tooth is trapezoidal, although other contemplated embodiments utilize other tooth shapes, such as but not limited to semicircular, rectangular, polygonal, and triangular. FIG. 2d is a top plan view of one embodiment of a reservoir chamber-cover assembly seal, illustrating a sealing tooth, a vacuum chamber, and an inlet and outlet chamber. FIG. 2e-1 is a cross-sectional view of one embodiment of a cover assembly seal connected to a reservoir, illustrating a cover gasket and a sealing tooth. FIG. 2e-2 is an enlarged view of a portion (circled) of FIG. 2e-1. As a pressure manifold (discussed below) engages the cover assembly, pressure forces the cover assembly (including the cover gasket) into the sealing tooth, forming a seal between each of the reservoir chambers.
FIG. 3A depicts an embodiment of a microfluidic device or chip, showing two channels with inlet and outlet ports, as well as an (optional) vacuum port. FIG. 3B is a top-view schematic drawing of an alternative embodiment of a perfusion disposable or "pod" characterized by a transparent (or translucent) cover over a reservoir with a chip inserted therein. The chip can be seeded with cells and then placed into a carrier for insertion into the perfusion disposable. FIG. 3C is a schematic drawing of an assembled perfusion disposable embodiment identical to that depicted in FIG. 3B , except that the ports and cutouts on the cover assembly (over the inserted chip for visualization, imaging, etc.) are depicted. FIG. 3D is a schematic drawing of the same perfusion disposable embodiment of FIG. 3C , but unassembled to illustrate the relationship of the cover, reservoir, skirt, chip, and carrier.
FIG. 4a is a side view of an embodiment of a chip carrier (having a chip therein) approaching a side track of a skirt of an embodiment of a perfusion manifold assembly (not yet engaged), the carrier being aligned at an angle matching the angled front end portion of the side track, the carrier including a retaining mechanism configured as an upwardly protruding clip. Without being bound by theory, it is believed that the suitably large angle allows chip engagement without smearing or premature engagement of droplets present on the chip and/or perfusion manifold assembly during the insertion and alignment process. FIG. 4b is a side view of an embodiment of a chip carrier (having a chip therein) engaging a side track of a skirt of an embodiment of a perfusion manifold assembly (not yet connected to said assembly). FIG. 4C illustrates a side view of one embodiment of a chip carrier (having a chip therein) that is fully engaged with the side tracks of the skirt of an embodiment of a perfusion manifold assembly (not yet connected to said assembly) (arrows illustrate the direction of movement required for a snap fit, whereby the retaining mechanism is engaged to prevent movement). FIG. 4D illustrates a side view of one embodiment of a chip carrier (having a chip therein) that is removably connected to a perfusion manifold assembly, wherein the retaining mechanism is engaged to prevent movement. While removability and optionally reattachability are desirable for certain applications (e.g., to allow chip removal to allow for cell addition, imaging, or performing various assays), in alternative embodiments, the connection is not releasable. For example, adhesive layers, glues, and/or heat stakes may be used to provide a robust connection that may be difficult to remove or reattach. FIG. 4e is a schematic diagram illustrating one embodiment of a connection approach to a perfusion manifold, including 1) a sliding action (4e-1), 2) a pivot (4e-2), and 3) a snap fit (4e-3) to provide alignment and fluid connection in a single motion. 1) In the sliding step, a chip (or other microfluidic device) is inserted into a carrier that slides to align the fluid ports. 2) In the pivot step, the chip (or other microfluidic device) is pivoted until the ports come into fluid contact. 3) In the clip or snap fit step, the force necessary to provide a secure seal is provided.
FIG. 5 is a schematic diagram of one embodiment of a workflow (with arrows depicting the individual steps) in which a chip is connected (e.g., snapped) to a disposable perfusion manifold assembly (a "perfusion disposable"), the assembly is placed back into another assembly on a culture module, and the culture module is installed in an incubator. In alternative embodiments, the culture module can include features of an incubator (e.g., a heat source and/or a source of warm, humid air) to eliminate the need for a separate incubator. While the present invention contemplates a "disposable" embodiment, the components are (alternatively) reusable (e.g., for cost reasons). In a further embodiment of the workflow or method, the chip can be installed in a carrier, the carrier can be installed in a seeding guide (discussed and depicted below), cells can be seeded onto the chip, the carrier can be removed from the seeding guide, and the carrier can be engaged with the perfusion disposable (the remainder of which is the same as the workflow depicted in FIG. 5 ).
FIG. 6 illustrates one embodiment of a culture module having a removable tray having a plurality of assemblies (having connected chips) positioned thereon, each of the perfusion manifold assemblies held in the tray next to it having pressure points on their mating surfaces corresponding to ports on the covers of the perfusion manifold assemblies so that they can be brought together by the tray mechanism such that pressure can be applied via a pressure controller. This allows the tray mechanism to attach all of the perfusion manifold assemblies to the pressure or flow controller (whether by raising the tray or lowering it to meet the tray) in a single motion, thereby simultaneously connecting them.
FIG. 7 is a side schematic drawing of another embodiment of a culture module showing a platform for positioning a removable tray that moves upward to a bonding surface so that pressure can be applied via a pressure controller (not shown).
FIG. 8A is a schematic diagram of another embodiment showing a tray (or rack) and sub-trays (or nests) for transporting and inserting perfusion disposables (PDs) into a pressure module having user interfaces on the exterior of the housing. FIG. 8B is a schematic diagram of another embodiment showing a tray (or rack) inserted into a housing of a culture module having user interfaces. The nested design, where the trays are shown holding multiple removable sub-trays (as an example of the present invention), provides the user with the flexibility to remove or retain a variety of PDs depending on the application. For example, the user may transfer all trays to a biosafety cabinet to replenish media or collect samples from all PDs in the tray, or may move a sub-tray of three PDs to a microscope stage to image three PDs without de-temperature and gas content regulating the remaining PDs, or may remove or load a single PD for careful inspection or replacement.
FIG. 9A is a schematic drawing (open position) of the interior of one embodiment of a pressure module, wherein the trays (or racks), sub-trays (or nests), and perfusion disposables (PDs) are positioned below the pressure manifold (but not interlocked therewith, with sufficient clearance to allow for their removal), and the actuation assembly (comprising a pneumatic cylinder) is above the pressure manifold. While three microfluidic devices or perfusion disposables are shown, typically more (e.g., 6, 9 or 12) are used at a time.
FIG. 9b is a schematic diagram (enclosed position) of the interior of one embodiment of a pressure module, wherein the trays (or racks), sub-trays (or nests), and perfusion disposables (PDs) are positioned below the pressure manifold (in engagement therewith), and the actuation assembly (including the pneumatic cylinder) is above the pressure manifold. Again, while three microfluidic devices or perfusion disposables are shown, typically more (e.g., 6, 9 or 12) are used at a time.
FIG. 10a is a schematic drawing of one embodiment of a pressure manifold (50) illustrating a PD engagement surface (54) having several PD engagement locations (in this case, six engagement locations). FIG. 10b is an enlarged portion of the engagement surface (54) of the pressure manifold (50) highlighting the spring shuttle (55), the valve seal (56), and the alignment feature (57) (which aligns the PD with the manifold). FIG. 10c is a schematic drawing of another embodiment of the pressure manifold (50) illustrating the PD engagement surface (54) along an enlarged portion highlighting the shroud compressor (58), the valve seal (56), and the alignment feature (57) (which aligns the PD with the manifold). FIG. 10d is a side schematic drawing of one embodiment of the pressure manifold (50) illustrating the PD engagement surface (54). FIG. 10e is a schematic drawing of one embodiment of a pressure manifold (50) showing the opposing surface (67). FIG. 10f is a schematic drawing of one embodiment of a pressure manifold (50) showing the PD engagement surface (54) with the PD guide (68) and lower backer plate (69) removed, highlighted by showing one spring carrier (70) and spring (71) (among others) removed from the manifold body, and one seal (72), shuttle (73), and valve body (74) (among others) removed from the manifold body. An external spring (75) adapted to press the pressure manifold against a perfusion disposable is also highlighted by showing it removed. FIG. 10g is a schematic drawing of one embodiment of a pressure manifold (50) showing the opposing surface (67) (not the PD mating surface) with the upper backer plate (76) and capping strip (77) removed. A manifold passage channel (78) adapted to direct and optionally distribute pressure and/or fluid from one or more pressure ports is shown. Additionally, one screw (79) (of several) and one gas port (80) (of five including both gas and vacuum ports) are shown removed from the manifold body (50). FIG. 10h is a schematic drawing of one embodiment of a pressure manifold (50) showing a top view of the manifold passage channel (78) and one of the several ports (81).
FIG. 11a is a schematic drawing of one embodiment of a valve (59) (Schrader valve) in a pressure manifold (50), showing a silicone membrane (60), a shuttle (61), an air inlet (62), and a cover plate (63). FIG. 11b is a side view photograph, and FIG. 11c is a top view photograph, of one embodiment of a valve for the pressure manifold, showing a valve seat (64) and a membrane (60) acting as valve seals. FIG. 11d is a schematic drawing of the interior side view of one embodiment of the pressure manifold (50), showing a plurality of valves (59), a poppet (65), a valve seal (66), and a PD cover (11) in a manifold body. In operation (to engage the PD), the valve seal (66) is deflected by the displacement of the poppet (65).
FIG. 12a is a schematic diagram of one embodiment of a connection scheme including a tubing connection manifold (82) that allows four culture modules (30) (three shown) to be connected within a single incubator (31) using one or more hub modules (the two circles provide enlarged views of the first end (83) and the second end (84) of the connection). FIG. 12b is a photograph of a gas hub and a vacuum hub (collectively 85) along tubing (86) for the connection shown in FIG. 12a.
FIG. 13 is a photograph of one embodiment of an incubator (sealed from the outside by an outer door) containing a shelf (not shown) capable of supporting a perfusion manifold assembly of the present invention. The incubator can have automated liquid handling, imaging and sensing features for automated experiments, cell viability assessment and/or experimental result collection. In one embodiment, the microfluidic device is connected during incubation.
Figure 14 is a schematic diagram of one embodiment of connecting two microfluidic devices to introduce air bubbles into the microchannels. Figure 14a depicts two fluidically primed devices (fluid is shown as a meniscus) with ports and microchannels that are not yet connected. Figure 14b depicts the devices of Figure 14a contacted in such a way that air bubbles (air is shown midway between each meniscus) are introduced into the ports (and ultimately the microchannels).
Figure 15 is a schematic diagram of one embodiment of connecting two microfluidic devices (or a microfluidic device to a fluid source) using a drop-to-drop approach without creating air bubbles. Figure 15a illustrates two fluidically primed devices having microchannels with protruding droplets formed on the surfaces of the devices, more particularly directly on and above the ports, rather than in the region surrounding the fluid paths or ports. Figure 15b illustrates that the droplet surfaces join together, typically without introducing any air bubbles, when the surfaces are in close proximity to each other during connecting.
FIG. 16a illustrates an embodiment of a microfluidic device contacting a fluid source or another microfluidic device, wherein the microfluidic device is approached from the side. FIG. 16b illustrates an embodiment of a microfluidic device contacting a fluid source or another microfluidic device, wherein the microfluidic device is approached from the bottom side, creating a drop-to-drop connection that establishes fluid communication (FIG. 16c). FIG. 16d illustrates another approach of contacting a microfluidic device with a fluid source or another microfluidic device, wherein the microfluidic device pivots.
FIG. 17 is a schematic diagram illustrating a confined droplet (22) on a surface (21) of a microfluidic device (16) in a path or port.
FIG. 18 is a schematic diagram showing a confined droplet (22) on a surface (21) of a microfluidic device (16) in the region of a path or port, wherein the droplet rests on a molded pedestal or mount (42), covers the entrance to the port, and protrudes above the port, the port being in fluid communication with the microchannel.
FIG. 19 is a schematic diagram showing a confined droplet (22) on a surface (21) of a microfluidic device (16) in the region of a path or port, wherein the droplet is on a gasket (43), covers the entrance to the port, and protrudes above the port, the port being in fluid communication with the microchannel.
FIG. 20 is a schematic diagram showing a confined droplet (22) in the region of a path or port, where a portion of the droplet is positioned below a surface (21) of a microfluidic device (16), wherein the droplet is on a shaped recess or depression (44) and covers the entrance to the port, with a portion protruding above the surface, the port being in fluid communication with the microchannel.
FIG. 21 is a schematic diagram showing a confined droplet (22) in the region of a path or port, where a portion of the droplet is located below a surface (21) of a microfluidic device (16), wherein the droplet is within a surrounding gasket, covers the entrance to the port, and a portion protrudes above the gasket.
Figure 22 is a schematic diagram illustrating a surface modification embodiment. Figure 22a illustrates a droplet (22) that uses a hydrophilic adhesive layer or sticker (45) and spreads from an edge of the sticker and is bound by a surrounding hydrophobic surface. Figure 22b illustrates a droplet (22) that spreads on a hydrophilic surface of a device and is bound by a surrounding hydrophobic surface.
Figure 23 is a schematic diagram of a surface modification embodiment (indicated by the arrows drawn in the “‡” direction below) utilizing surface treatment with a mask (41).
FIG. 24 is a schematic diagram of one embodiment of a drop-to-drop connection scheme that controls droplets using a combination of geometrical features and surface treatments. FIG. 24a depicts an embodiment of a microfluidic device or "chip" comprising fluidic channels and ports having an elevated region (e.g., a pedestal or gasket) at each port. FIG. 24b depicts a hydrophilic channel filled with fluid where the droplet radii are balanced at each end (i.e., at the port openings). FIG. 24c depicts a portion of the microfluidic device of FIG. 24b having an upwardly protruding droplet (22) approaching (but not yet contacting) a portion of a mating surface of a perfusion manifold assembly having a protruding droplet (in this case, a droplet (23) protruding downwardly). Figure 24d shows the same portion of the microfluidic device of Figure 24c with the upwardly protruding droplet (22) of the microfluidic device in contact with (fused with) the downwardly protruding droplet (23) of the perfusion manifold assembly.
Figure 25 illustrates an embodiment of a drop-to-drop connected using only surface treatment (i.e., without a geometry such as a pedestal or gasket). Figure 25a illustrates an embodiment of a perfusion manifold assembly including fluid channels and ports. Figure 25b illustrates a hydrophilic channel filled with fluid to a predetermined level (e.g., to the height of a fluid column).
Figure 26 is a chart showing (without being bound by theory) the relationship between the port diameter (in millimeters) and the maximum hydrostatic head (in millimeters) that a stabilized drop can support.
FIG. 27 illustrates an embodiment in which a microfluidic device (“chip”) is connected from below to a flow manifold assembly (top) at a port having an exhaust gasket (43), wherein the assembly does not cover or seal the gasket, allowing any air entrapped during connection to be exhausted (right arrow). It may be desirable to ensure that any air preferentially flows out through the exhaust gasket rather than continuing to flow through the channels. In some embodiments, this preferential flow can be facilitated by applying a first pressure (P1) to the fluid in the fluid channels of the assembly (left arrow) and applying a second pressure (P2) to the fluid in the microfluidic device channels, wherein P1 and P2 are greater than the back pressure of the exhaust gasket. In some embodiments, the pressures P1 and/or P2 are applied using a pressure source and/or gravitational head. In some embodiments, the pressures P1 and/or P2 are generated by flow resistance of the fluid.
FIG. 28 illustrates another embodiment where the microfluidic device (16) (“chip”) is connected from below to the perfusion manifold assembly (10) (above) at a port having an exhaust gasket (43), wherein the assembly covers the gasket (i.e., the gasket is surrounded by the assembly mating surfaces), but there is a passage in the assembly above the gasket to allow any air trapped during the connection to be exhausted.
FIG. 29 illustrates another embodiment where the microfluidic device (16) (“chip”) is connected from below to the flow manifold assembly (10) (top) at a port having an exhaust gasket (43), wherein the fluid passage passes over the gasket (the gasket could be larger if desired). This embodiment facilitates removal of trapped air, such as smaller bubbles, during connection, and without being bound by theory, this is because it allows the smaller bubbles to interact (“wet”) with the exhaust gasket.
FIGS. 30 and 31 are a series of still photographs from a video showing an embodiment of a microfluidic device (having a droplet protruding from a gasket) moving along an essentially linear rail or guide track of a fluid source, or microfluidic device, such as a perfusion manifold assembly (i.e., along the x-axis in the x/y plane) until approaching a corresponding port of a perfusion manifold assembly (compare FIGS. 30a and 30b ), wherein a combination of x- and z-axis movement (i.e., lateral movement and upward movement) fuses the droplets and connects the chip ( FIG. 31b ).
FIG. 32a illustrates one embodiment of a fluid backplane including a meandering fluid resistor channel (91), a vacuum channel (92), and a drain channel (93). FIG. 32b is an edge view. FIG. 32c illustrates a chip engagement relief (94) of the fluid backplane that acts as a fluid drain port, along with an assembly alignment feature (95) and a visualization cutout (96) that allows for microscopic observation and other imaging.
Figure 33 illustrates a schematic of an exemplary microfluidic device or “organ-on-a-chip” device. The assembled device is schematically illustrated in Figure 33a. Figure 33b illustrates an exploded view of the device of Figure 33a.
Figure 34 is a schematic diagram illustrating an embodiment having two membranes.
FIG. 35a illustrates components of one embodiment of an unassembled culture stand or holder (100) including first and second end caps (106 and 107) and first and second side panels (108 and 109). FIG. 35b illustrates a chip (16) and a carrier (17) within a seeding guide with the seeding guide approaching (but not engaging) the stand (100). FIG. 35c illustrates six seeding guides including a carrier (17) (having chips) mounted on the stand (100).
FIGS. 36a-c are photographs of an embodiment of a perfusion manifold assembly lacking a skirt (or other protrusion) with side tracks for engaging a chip (or other microfluidic device) in a carrier. Instead, the base (110) of the assembly (10) is configured to receive the carrier (17) from below (instead of from the side) in a Lego™ block type connection, i.e., the base (110) has a cavity (111) and an opening (112) dimensioned to receive the carrier (17), while a handle or tab (18) of the carrier is configured to fit into the opening (112). FIG. 36a is a top side view of the assembly (10) prior to engaging the carrier (17) and the chip (16). Figure 36b is a bottom side view of the assembly (10) having a fluid discharge port (94) configured to align with a port (2) on the chip (16). Figure 36c shows the assembly (10) engaged with the carrier such that the carrier tab (18) is positioned in the opening (112).

definition

"Binding number" is a dimensionless ratio of gravity to capillary forces on a liquid interface. The higher the bonding number of air, the more likely it is that liquid interfaces will be formed by gravity. The lower the bonding number, the more likely these surfaces will be formed by capillary forces.

A "hydrophobic coating" may be formed on a surface (or portion thereof), such as a protrusion, platform or pedestal at or near a port, as well as on a bonding surface (or portion thereof), using a "hydrophobic agent." It is not intended that the present invention be limited to a particular hydrophobic agent. In one embodiment, the present invention contemplates forming a hydrophobic coating using silanes, such as, but not limited to, halogenated silanes and alkylsilanes. In this regard, the present invention is not intended to be limited to a particular silane; the choice of silane is limited solely from a functional standpoint, i.e., which renders the surface hydrophobic. The present invention also contemplates using commercially available products, such as the Rain-X™ product (a synthetic hydrophobic surface-applied product that beads up water, most commonly used on glass automotive surfaces).

A surface or region on a surface is "hydrophobic" if it has an advancing contact angle for water (e.g., greater than about 90°) (in many cases, it is preferred that both the advancing and receding contact angles be greater than about 90°). In one embodiment, the hydrophobic surface of the present invention exhibits an advancing contact angle for water of from about 90° to about 110°. In another embodiment, the hydrophobic surface has a region that exhibits an advancing contact angle for water of greater than about 110°. In another embodiment, the hydrophobic surface has a region that exhibits a receding contact angle for water of greater than about 100°. It is important to note that some liquids, particularly some biological liquids, may contain components that can coat a surface after it has been wetted with it, thereby affecting its hydrophobicity. In the context of the present invention, it may be important that the surface resist such coating from the intended use liquid, such that such coating does not produce advancing and/or receding contact angles of less than 90° while the surface is wetted with said liquid.

A surface or region on a surface is "hydrophilic" if it has a (e.g., advancing) contact angle with water of less than about 90°, more commonly less than about 70° (in many cases, it is desirable for both the advancing and receding contact angles to be less than about 90° or less than about 70°).

The measured contact angle can be within a given range, i.e., the range of the so-called advancing (maximum) contact angle to the receding (minimum) contact angle. The equilibrium contact lies within these values and can be calculated from them.

Hydrophobic surfaces "resist wetting" by aqueous liquids. When the contact angle formed by a first liquid on a material is greater than 90°, the material is said to be resistant to wetting by the first liquid. A surface can be resistant to wetting by aqueous liquids and non-aqueous liquids, such as oils and fluorinated liquids. Some surfaces can be resistant to wetting by both aqueous and non-aqueous liquids. Hydrophobic behavior is generally observed by surfaces having a critical surface tension less than 35 dyne/cm. First, a decrease in the critical surface tension is associated with oleophilic behavior, i.e., wetting of the surface by hydrocarbon oils. As the critical surface tension decreases below 20 dyne/cm, the surface resists wetting by hydrocarbon oils and is considered to be hydrophobic as well as oleophobic.

A hydrophilic surface "facilitates wetting" by an aqueous liquid. A material is said to facilitate wetting by a first liquid if the contact angle formed by the first liquid on the material is less than 90°, more commonly less than 70°.

As used herein, the phrases "connected," "connected to," "coupled to," "in contact with," and "in communication with," refer to any form of interaction between two or more entities, such as mechanical, electrical, magnetic, electromagnetic, fluidic, and thermal interactions. For example, in one embodiment, a channel in a microfluidic device is in fluidic communication with a cell and (optionally) a fluid reservoir. The two components can be coupled to each other even if they are not in direct contact with each other. For example, the two components can be coupled to each other via an intermediate component (e.g., tubing or other conduit).

A "channel" is a path (straight, curved, single, multiple, network, etc.) through a medium (e.g., silicon, plastic, etc.) that allows the movement of liquids and gases. Thus, a channel can connect other components, i.e., keep the components "in communication", more particularly "in fluid communication", even more particularly "in liquid communication". Such components include, but are not limited to, liquid-inlet ports and gas-outlet ports.

A "microchannel" is a channel having a dimension less than 1 millimeter and greater than 1 micrometer. Additionally, the term "microfluidic" as used herein refers to a component that confines or directs fluid movement through one or more channels having one or more dimensions of 1 mm or less (microscale). The microfluidic channels can be larger than microscale in one or more directions, but the channel(s) will be microscale in at least one direction. In some examples, the geometry of the microfluidic channels can be configured to control the fluid flow rate through the channels (e.g., to increase channel height to reduce shear). The microfluidic channels can be formed in a variety of geometries to facilitate a wide range of flow rates through the channels.

The present invention contemplates a variety of "microfluidic devices", including but not limited to microfluidic chips (e.g., as illustrated in FIG. 3A ), perfusion manifold assemblies (chipless), and perfusion manifold assemblies interdigitated with microfluidic chips (e.g., as illustrated in FIG. 3B ). However, the methods described herein for interdigitating microfluidic devices (e.g., by drop-to-drop connections) and perfusing the microfluidic devices are not limited to any particular embodiment of the microfluidic devices described herein, and may generally be applied to microfluidic devices, such as devices having one or more microchannels and ports.

A "stable drop" is a drop of medium that does not experience significant movement (e.g., remains in contact with a fluid port) from its intended location, preferably over a period of several seconds, more preferably over a period of one minute, and even more preferably over several minutes (2-10 minutes), and does not experience significant (>10%) change in volume or configuration on the microfluidic device. In a preferred embodiment, the present invention contemplates a stable drop during drop-to-drop engagement. A surface may be inherently (e.g., by virtue of its construction) capable of holding a droplet stable, or may be made to hold a droplet stable, meaning that the droplet will not spontaneously expand or move beyond a confined (or designated) region. A stable droplet does not experience significant change in volume or configuration. The present invention contemplates such spatial control of the droplet, i.e., holding the droplet within a confined spatial extent, and/or holding the droplet within a spatial extent of one or more regions. In a preferred embodiment, the present invention contemplates preventing the droplet from extending too far, and ensuring that the droplet remains centered in the port (i.e., ensuring that the area directly above the fluid port remains covered by the droplet). In terms of preventing the droplet from extending or spreading too far, the present invention contemplates, in one embodiment, maintaining the droplet within the spatial extent of one or more regions. In a particularly preferred embodiment, the present invention contemplates preventing the droplet from moving during manipulation (i.e., rolling around on the surface as the microfluidic device or chip is moved around or even flipped over). Of course, such movement is contemplated without vigorous shaking. A droplet that is found to be stable when a particular engagement procedure is utilized may be found to be unstable when another procedure (e.g., a more vigorous procedure) is utilized.

“Controlled engagement” means proper alignment and flatness of the paths or ports. Refers to the interlocking of two devices that allows for both a point-to-point connection and does not result in a loss of point stability. For example, if the devices are rigidly fixed in place or meet before engaging the point on the opposing device, the point stability will be compromised.

General description of the invention

In one embodiment, the present invention contemplates a perfusion manifold assembly (preferably removably) connected to a microfluidic device, such as an organ on a chip microfluidic device comprising cells that mimic cells present in an organ in the body or that mimic at least one function of an organ, such that the perfusion manifold assembly allows fluid to flow from a fluid reservoir into a port of the microfluidic device, optionally without tubing, at a controllable flow rate. In one embodiment (as illustrated in FIGS. 1a, 1b, and 1c), a perfusion manifold assembly (10) comprises i) a cover or lid (11) configured to act as an upper portion of one or more fluid reservoirs, ii) one or more fluid reservoirs (12), iii) a capping layer (13) disposed beneath said fluid reservoir(s), iv) a fluid backplane (14) comprising a resistor, and in fluid communication with said fluid reservoir(s) beneath said fluid reservoir(s), and v) a protrusion or skirt (15) for engaging with a microfluidic device (16) or chip, preferably positioned in a carrier (17), said chip having one or more microchannels (1) and in fluid communication with one or more ports (2). The assembly can be used with or without the lid or lid. Other embodiments (discussed below) lack the skirt or protrusion. In one embodiment, the carrier (17) includes a tab or other gripping platform (18), a retention mechanism, such as a clip (19), and a visualization cutout (20) for imaging the chip. The cutout (20) allows the carrier (e.g., engaged or not with a perfusion manifold assembly or “pod”) to be mounted on a microscope or other inspection device, allowing the chip to be viewed without having to remove the chip from the carrier. In one embodiment, the fluidic resistor includes a series of switchback or tortuous fluidic channels. FIG. 32 depicts an improved schematic of one embodiment of a backplane, illustrating the fluidic resistor channels (32a) and the chip engagement reliefs (32c) or ports. Various fluid resistor designs are contemplated, such as those more fully described in U.S. Provisional Patent Application Serial Nos. 62/024,361 and 62/127,438 (PCT/US2015/040026, incorporated herein by reference, particularly with respect to the discussions of resistors, resistor design, and pressure). In one embodiment, the perfusion manifold assembly is made of plastic and is disposable, i.e., disposed of after docking to and perfusing the microfluidic device. While the present invention contemplates a "disposable" embodiment, the components are (alternatively) reusable (e.g., for cost considerations).

In one embodiment, the microfluidic device (e.g., chip) (16) may be first mounted in a carrier (17) (e.g., a chip carrier) prior to being mated with the perfusion manifold assembly (10), or may be mated directly with the assembly. In either case, the (optionally) releasable connection of the microfluidic device to the manifold should either a) prevent air from entering the microchannels, or b) provide a way for undesirable air to be removed or vented from the system. Indeed, air removal may be required both during chip attachment and during use of the microfluidic device in some embodiments.

In one embodiment to prevent air from entering the microchannels, the microfluidic devices are releasably connected using a "drop-to-drop" "chip-to-cartridge" connection. In this embodiment, the injection port of the microfluidic device has a droplet (22) protruding therefrom ( FIG. 15a ), and the surface of a perfusion manifold assembly or "cartridge" (10) for engagement with the device has a corresponding droplet (23). After the two come together ( FIG. 15b ), the drops fuse, allowing fluid communication without introducing air into the channels. In one embodiment, the chip carrier is designed so as not to interfere with the "drop-to-drop" connection. For example, the carrier surrounds the side of the microfluidic device in one embodiment, but does not surround its mating surface (21). Note that FIG. 15a depicts a perfusion manifold (10) without a skirt, and that the microfluidic device or chip is engaged from below (rather than from the side) of the perfusion manifold.

It is not intended that the present invention be limited to only one way in which microfluidic devices can be releasably connected. In one embodiment, a microfluidic device, such as an organ-on-a-chip microfluidic device that mimics one or more functions of cells in an organ of the body, or that includes cells that mimic at least one function of an organ, is accessed from the side (FIG. 16A) or from below (FIG. 16B) such that a droplet (22) protrudes upward, while a corresponding droplet (23) on the assembly (or other type of fluid source) protrudes downward. The microfluidic device (or device carrier) may include a portion (24) configured to engage a side track (25) or other guide mechanism. In another embodiment, a microfluidic device, such as an organ-on-a-chip microfluidic device that mimics cells in an organ of the body, or that includes cells that mimic at least one function of an organ, is accessed from the top such that a droplet protrudes downward, while a corresponding droplet on the assembly protrudes upward. In yet another embodiment, a microfluidic device, such as an organ-on-a-chip microfluidic device comprising cells that mimic cells found in an organ of the body or that mimic at least one function of an organ, approaches the assembly from the side and is positioned by a pivot (see FIG. 16d , arrow) at a hinge, socket, or other pivot point (26). In yet another embodiment, the microfluidic device is engaged in the manner of an audio cassette or CD such that the droplets protrude upward, while the corresponding droplets on the assembly protrude downward, wherein the lateral and upward movements are combined (FIGS. 16b-16c).

In one embodiment, the microfluidic device (16) is removably connected to the perfusion manifold assembly (10) by a clipping mechanism that temporarily "locks" the microfluidic device, such as an organ device on a chip, in place (FIGS. 4A, 4B, 4C and 4D). In one embodiment, the clipping or "snap fit" involves protrusions on the carrier (19) that act as a retaining mechanism when positioning the microfluidic device (16). In one embodiment, the clipping mechanism is similar to the interlocking plastic design of LEGO™ chips and includes straight-down clips, friction fit, radial-compression fit, or a combination thereof. However, in another embodiment, the clipping mechanism is triggered only after the microfluidic device, or more preferably the carrier (17) comprising the microfluidic device (16), has engaged the perfusion manifold assembly (or cartridge) on the guide rail, side slot, inner or outer track (25), or other mechanism that provides a stable gliding path for the device as it is moved into position (e.g., by machine or by hand). The guide rail, side slot, inner or outer track (25) or other mechanism may be strictly linear, but is not necessarily so, and may be located on a protruding member or skirt (15) attached to the main body of the perfusion manifold assembly (10). In one embodiment, the starting portion of the guide rail (25) (or side slot, inner or outer track or other mechanism) comprises a linear or essentially linear portion (28) followed by an angled slide (27) that provides a larger opening for easier initial positioning. In one embodiment, the distal portion (29) (proximate the corresponding port in the assembly) of an otherwise linear (or essentially linear) guide rail (25) (or side slot, inner track or other mechanism) is angled upward (or curved) (FIG. 16b) to achieve connection by combining linear (e.g., initial) and upward movement.

In some embodiments, it is important that the droplets remain in place in their corresponding fluid ports despite any period of movement or inversion of their substrate. It is also desirable that the droplets maintain their size, for example, so that the drop-to-drop process is constant regardless of the speed of the engagement process. Accordingly, the present invention contemplates designs and methods that provide stable droplets. Stable droplets are contemplated for aqueous as well as non-aqueous liquids. While the inventors have focused on embodiments of the present invention without loss of generality for aqueous droplets, those familiar with the art will be able to adapt the embodiments, particularly the use of hydrophilic and hydrophobic regions or materials based on the wetting properties of the liquid. In some embodiments, the droplet can be confined to a first region of the substrate by surrounding the first region with a second region, wherein the second region is hydrophobic (or more generally tends to resist wetting of the droplet by the liquid). The second region can be rendered hydrophobic by selection of one or more hydrophobic materials included (e.g., PTFE, FEP, certain grades of nylon, etc.), a surface treatment (e.g., a plasma treatment, a chemical treatment, an ink treatment), use of a gasket (e.g., a film, an o-ring, an adhesive gasket), masking during the treatment of at least one of the other regions of the substrate, or a combination thereof. In some embodiments, the first region can be confined to the first region of the substrate by surrounding the first region with geometric features. In some embodiments, the geometric features can be elevations or depressions. Without being bound by theory, such features can act to confine the droplet by utilizing their edges to interact with a surface layer of the droplet (and correspondingly with the surface tension of the droplet), for example, by "pinning" the surface of the droplet. In some embodiments, the droplet can be restricted to cover a first region of the substrate by adapting the first region to be hydrophobic (or more generally, prone to wetting by the liquid of the droplet). The first region can be rendered hydrophilic by selection of one or more hydrophilic materials included (e.g., PMMA, PLA), a surface treatment (e.g., plasma treatment, chemical treatment, ink treatment), use of a gasket (e.g., a film, o-ring, adhesive gasket), masking during treatment of at least one other region of the substrate, or a combination thereof.

In one embodiment, the bonding surface (21) of the microfluidic device (or at least a portion thereof adjacent the port opening) is hydrophobic or is rendered hydrophobic (or is masked to prevent it from becoming hydrophilic during the plasma treatment). In one embodiment, the bonding surface of the perfusion manifold assembly or cartridge (or at least a portion thereof adjacent the port opening) is hydrophobic or is rendered hydrophobic (or is masked to prevent it from becoming hydrophilic during the plasma treatment). In one embodiment, both the bonding surface of the microfluidic device (or at least a portion thereof adjacent the port opening) and the bonding surface of the perfusion manifold (or at least a portion thereof adjacent the port opening) are hydrophobic or are rendered hydrophobic (or is masked to prevent it from becoming hydrophilic during the plasma treatment).

An advantage of the carrier is that it need not contact the surfaces of the microfluidic device during its releasable connection with the perfusion manifold assembly. The carrier may have a plate, platform, handle or other mechanism for gripping the carrier (18) without contacting the mating surface (21) of the microfluidic device (16). The retaining mechanism (19) may include a protrusion, hook, latch or lip that engages with one or more portions of the perfusion manifold assembly, and more preferably the skirt of the perfusion manifold assembly, to provide a "snap fit."

In other embodiments (FIGS. 27, 28, and 29), one or more gaskets can be used to vent air (e.g., any air introduced due to the releasable connection of the microfluidic device to the perfusion manifold assembly). In one embodiment, bubbles can be captured (and thus their impact limited), and in alternative embodiments, bubbles can be vented. One approach involves using a hydrophobic venting material (either molded or sheet). For example, the hydrophobic venting material can comprise PTFE, PVDF, hydrophobic grades of nylon, or combinations thereof. In some embodiments, venting can be accomplished by using a material that exhibits high gas permeability (e.g., PDMS). In other embodiments, venting can be accomplished by using a porous material, such as a sintered material, a porous membrane (e.g., track-etched membrane, fiber-based membrane), an open-celled foam, or combinations thereof. In a preferred approach, air is released from the vented (or venting) gasket. In some embodiments, the perfusion manifold assembly or microfluidic device includes an exhaust port adapted to provide a passage through which unwanted gases are released.

Once the microfluidic devices (or "chips") are docked to the perfusion manifold assembly, the assembly-chip combination can be installed in an incubator (31) (typically set to a temperature greater than room temperature, e.g., 37°C), or more preferably, in a culture module (30) capable of supporting multiple assembly-chip combinations, said culture module being configured to fit into an incubator shelf (see FIG. 5 ). This allows for easy handling of multiple (e.g., 5, 10, 20, 30, 40, 50, or more) microfluidic devices at one time. For example, where the culture module comprises nine assembly-chip combinations and the incubator is sized for six to nine culture modules, then from 54 to 81 "organs-on-chip" can be handled in a single incubator ( FIGS. 5 and 8 ). In another example, where the culture module comprises 12 assembly-chip combinations and the incubator is sized for 4 to 6 culture modules, 48 to 72 "organs-on-a-chip" can be handled in a single incubator. The perfusion manifold can be easily removed and inserted into the culture module without disrupting the fluidic connections to the chip. In one embodiment, the culture module can be maintained at a temperature greater than room temperature, for example, 37° C., even when not placed in an incubator.

In one embodiment (FIG. 6), the culture module (30) includes a removable tray (32) for positioning the assembly-chip assembly, a pressure surface (33), and optional user interfaces (46) for controlling movement of the various elements, together with a pressure controller (34). In one embodiment, the tray (32) is slidable. In one embodiment, the tray is positioned on the culture module and the tray is moved upwardly via the tray mechanism (35) such that it engages the pressure surface (33) of the culture module, i.e., the cover or lid (11) of the perfusion manifold assembly (10) engages the pressure surface of the culture module (30). Multiple perfusion assemblies (10) can be attached to the pressure controller by a single motion by the tray mechanism. In another embodiment, the tray is positioned on the culture module and the pressure surface of the culture module (30) is moved downward such that it engages the tray (32), i.e., the cover or lid (11) of the perfusion manifold assembly (10). In either case, in one embodiment (FIGS. 2A and 2B), the cover or lid includes ports, such as through-hole ports (36), that engage with corresponding pressure points on the pressure surface (33) of the culture module. These ports (36) transmit the applied pressure internally through the cover and through the gasket (37) when engaged, thereby applying pressure to the fluid in the reservoirs (12) of the perfusion manifold assembly (10). Thus, in this embodiment, pressure is applied to the reservoirs (12) through the cover (11) and the lid seal. For example, when 1 kPa is applied, this nominal pressure produces a flow rate of approximately 30-40 uL/hr in one embodiment. Alternatively, these ports (36) move inwardly on the cover so as to contact the gasket when engaged (i.e., the ports essentially act like plungers).

FIG. 8a is a schematic drawing of another embodiment of a culture module (30) showing trays (or racks) (32) and sub-trays (or nests) for transporting and inserting perfusion disposables (10) into the culture module, the culture module having two openings (48, 49) in the housing for receiving the trays, and a user interface (46) for controlling the process of engaging and pressurizing the perfusion disposables. A typical incubator (not shown) can support up to six modules (30). FIG. 8b is a schematic drawing of the same embodiment as FIG. 8a, but with two trays (or racks) (32) inserted into two openings (48, 49) in the housing (53) of the pressure module (30) which has a user interface (46) (e.g., an LCD screen) for process control.

FIG. 9a is a schematic diagram of the interior of one embodiment of a module (i.e., with the housing removed) wherein the pressure manifold (50) is in the open position, the tray or rack (32), the sub-tray or nest (47), the perfusion disposable (10) is positioned below the pressure manifold (50) but not engaged therewith (with sufficient clearance to allow for their removal), and the actuating assembly (51) includes the pneumatic cylinder (52) thereabove.

FIG. 9b is a schematic diagram of the interior of one embodiment of a module (i.e., with the housing removed) wherein the pressure manifold (50) is in a closed position, the trays or racks (32), the sub-trays or nests (47), the perfusion disposables (10) are positioned below and engaged with the pressure manifold (50), and the actuation assembly (51) includes a pneumatic cylinder (52) thereon. The pressure manifold (50) simultaneously engages all of the perfusion disposables (10) while media perfusion is desired or needed. Independent control of flow rates in the upper and lower channels of the chip (16) can be achieved. The pressure manifold (50) can be detached (without complex fluid separation) to remove the trays (32) or nests (47) for imaging or other operations, as needed. In one embodiment, the pressure manifold (50) can be detached from multiple perfusion manifold assemblies simultaneously. In one embodiment, the perfusion disposables (10) can be positioned in proximity to the pressure manifold (50) without being rigidly secured to the nest (47). In a preferred embodiment, the alignment features incorporated in the pressure manifold (50) provide a guide for each perfusion disposable (10).

In one embodiment, the cover or lid is made of polycarbonate. In one embodiment, each through-hole port is associated with a filter (38) (e.g., a 0.2 um filter). In one embodiment, the filter is aligned with a hole (39) in a gasket located beneath the cover.

A culture module is contemplated that includes a pressure manifold that enables perfusion and optionally mechanical actuation of one or more microfluidic devices, e.g., organ microfluidic devices on a chip that include cells that mimic at least one function of an organ in the body. FIG. 10A is a schematic diagram of one embodiment of a pressure manifold (50) showing a PD engagement surface (54) having several PD engagement locations (in this case six engagement locations). FIG. 10B is an enlarged portion of the engagement surface (54) of the pressure manifold (50) highlighting a spring shuttle (55), a valve seal (56), and an alignment feature (57) (which causes the PD to align with the manifold). The spring shuttle is an optional means by which the pressure manifold can sense the presence of a PD at a particular PD engagement location. In certain embodiments, when the PD is present, the spring shuttle is depressed, causing the shuttle to open one or more valves located within the pressure manifold, thereby allowing the application of pressure or fluid flow to the PD. Additionally, when the PD is absent, the shuttle is not depressed, and the valves remain sealed; this is intended to prevent pressure or fluid leakage. The illustrated valve seals are adapted to form a pressure and/or fluid seal with respect to corresponding features in the PD and, if present, the pressure covers. FIG. 10C is a schematic drawing of another embodiment of a pressure manifold (50), illustrating the PD engagement surface (54) along an enlarged portion highlighting the cover compressor (58), the valve seal (56), and the alignment feature (57) (which aligns the PD with the manifold). The cover compressor can apply a force on the pressure cover to assist in establishing a pressure and/or fluid seal between the pressure cover and the reservoir. In one embodiment, the cover compressor comprises a spring, an elastomeric material, a pneumatic actuator, or a combination thereof, which may be selected and sized to apply a force commensurate with the force necessary to maintain the pressure and/or fluid seal. FIG. 10d is a side schematic drawing of one embodiment of a pressure manifold (50), illustrating a PD engagement surface (54). FIG. 10e is a schematic drawing of one embodiment of a pressure manifold (50), illustrating an opposing surface (67). FIG. 10f is a schematic drawing of one embodiment of a pressure manifold (50) showing the PD engagement surface (54) with the PD guide (68) and lower backer plate (69) removed, with one spring carrier (70) and spring (71) (among others) removed from the manifold body, and one seal (72), shuttle (73), and valve body (74) (among others) removed from the manifold body. An external spring (75) adapted to press the pressure manifold against a perfusion disposable is also highlighted by being shown removed. FIG. 10g is a schematic drawing of one embodiment of a pressure manifold (50) showing the opposing surface (67) (not the PD engagement surface) with the upper backer plate (76) and capping strip (77) removed. A manifold pass-through channel (78) adapted to induce and optionally distribute pressure and/or fluid from one or more pressure ports is depicted. Additionally, one screw (79) (of several) and one gas port (80) (of five, including both gas and vacuum ports) are depicted as being removed from the manifold body (50). FIG. 10h is a schematic diagram of one embodiment of a pressure manifold (50) illustrating a top view of the manifold pass-through channel (78) and one of the several ports (81). The pass-through channels can be created using numerous methods known in the art, such as molding, machining, ablation, lamination, 3D printing, photolithography, and combinations thereof.

FIG. 11a is a schematic drawing of one embodiment of a valve (59) (Schrader valve) in a pressure manifold (50), showing a silicone membrane (60), a shuttle (61), an air inlet (62), and a cover plate (63). In this embodiment, a spring shuttle is incorporated into the valve and is adapted to actuate the valve by depressing a poppet of the Schrader valve. FIG. 11b is a side view photograph of one embodiment of a valve for a pressure manifold, showing a valve seat (64) and a membrane (60) acting as a valve seal, and FIG. 11c is a top view photograph thereof. FIG. 11d is a schematic drawing of the interior side of one embodiment of a pressure manifold (50), showing a plurality of valves (59), a poppet (65), a valve seal (66), and a PD cover (11) in a manifold body. When operating (to engage with the PD), the valve seal (66) is deflected by the displacement of the poppet (65).

FIG. 12a is a schematic diagram of one embodiment of a connection scheme including a tubing connection manifold (82) that allows four culture modules (30) (three shown) to be connected within a single incubator (31) using one or more hub modules (the two circles provide enlargements of the first end (83) and the second end (84) of the connection). FIG. 12b is a photograph of a gas hub and a vacuum hub (collectively 85) along tubing (86) for the connection shown in FIG. 12a. While this connection scheme is optional, it provides a convenient way to use multiple culture modules in a single incubator.

Detailed description of the invention

A. Pressure cover

The present invention contemplates, in one embodiment, a "perfusion manifold assembly" or "perfusion disposable" that facilitates culturing of organs on a chip within a culture apparatus. While the present invention contemplates a "disposable" embodiment, the component may (alternatively) be reused (e.g., for cost reasons).

In one embodiment, these perfusion disposables (PDs) comprise one or more inlets and one or more outlet reservoirs, as well as the necessary components for pumping. In particular, in embodiments of the present invention, the perfusion disposables comprise one or more resistors used for pressure-driven pumping (see FIG. 32a ). In pressure-driven embodiments, the device generates or controls fluid flow by applying pneumatic pressure (whether positive or negative) to one or more reservoirs. One advantage of this approach is that the pressure-driven design can avoid liquid contact with the device, which provides benefits in terms of sterility and ease of use (e.g., avoiding gas bubbles in the liquid lines). In some embodiments, the device applies pressure directly to one or more reservoirs (without a cover). A sufficient pressure seal can be achieved by one or more gaskets integrated into the perfusion disposable and/or the device (e.g., as part of a pressure manifold). However, when the perfusion disposable is external to the device, it is desirable for the reservoir to be protected from contamination, such as from environmental particles or airborne microorganisms. Thus, in the same embodiment, it may be desirable to utilize a PD embodiment that provides a cover that can be used by the user to cover the reservoir when external to the device and/or includes a substrate that transmits pressure but blocks contaminants (e.g., a suitable filter positioned over the opening of the reservoir). However, such solutions typically have drawbacks. In particular, when the user is expected to install the cover, it requires the user to manage the cover while the perfusion disposable is engaged with the device, and ideally to install the cover as soon as the PD is disengaged from the device, actions that in most situations negatively impact the user experience. In addition, a filter positioned over the opening of the reservoir typically blocks access to the reservoir by pipettes and other typical laboratory tools, negatively limiting their ease of use.

In accordance with an aspect of the present invention, the inventors disclose a "pressure cover" that can be positioned over a microfluidic device or a device adapted to receive a microfluidic device (e.g., a perfusion disposable) even while the device is engaged with the device, the pressure cover being adapted to allow pressure communication between the device and the device. The present invention contemplates in some embodiments that the pressure cover is a removable cover adapted to be positioned over one or more reservoirs of a microfluidic device or a device adapted to receive a microfluidic device (e.g., a perfusion disposable), the pressure cover comprising at least one device-facing port and at least one reservoir-facing port, the pressure cover being adapted to transfer pressure between at least a portion of the device-facing port and at least a portion of the reservoir-facing port. In some embodiments, the pressure cover comprises at least one "through hole" port, which is an opening connecting first and second surfaces of the cover, wherein the opening on the first surface is adapted to form an equipment-facing port, and the opening on the second surface is adapted to form a reservoir-facing port. In some embodiments, the through-hole port is circular, rectangular, triangular, polygonal, straight, curved, oval, and/or curved. However, in some embodiments, the cover comprises a channel connecting at least one equipment-facing port and at least one reservoir-facing port, wherein the ports are not arranged to directly face each other. Such embodiments may be useful, for example, where it is necessary to accommodate equipment connection locations and reservoir locations, for example, where the same equipment is desired to support operation of multiple types of perfusion disposables.

In some embodiments, the pressure cover is adapted to form a pressure seal between the pressure cover and at least one reservoir. In some embodiments, the pressure cover is engaged with at least one reservoir forming a cover-to-reservoir pressure seal. In some embodiments, the pressure cover is adapted to form a pressure seal between the pressure cover and at least one piece of equipment. In some embodiments, the pressure cover is engaged with at least one piece of equipment forming a cover-to-equipment pressure seal. Any of the cover-to-reservoir seals and cover-to-equipment seals can utilize any sealing method known in the art, and can be selected from the group consisting of face seals, radial seals, tapered seals, friction seals, or combinations thereof. Any of the seals can utilize one or more gaskets, o-rings, elastomeric materials, flexible materials, adhesives, sealants, greases, or combinations thereof. It is not intended that the present invention be limited to designs having a complete pressure seal, as a complete pressure seal may not be required. Rather, some amount of gas leakage can be tolerated because the equipment can actively control pressure to compensate for leakage. The requirement to achieve a complete seal on one or both sides can be relaxed, simplifying the design and reducing cost.

In some embodiments, the pressure cover comprises a load concentrator. For example, in some embodiments, the pressure cover comprises a ridge surrounding at least one equipment-facing port. In some embodiments, the pressure cover comprises a ridge surrounding at least one reservoir-facing port. It is known in the art that such load concentrators can act to improve pressure sealing by improving reliability or reducing required forces, and designs known in the art include, for example, rectangular, semicircular, triangular, trapezoidal, and polygonal ridges. Thus, a load concentrator surrounding an equipment-facing port can be used to improve cover-to-equipment pressure sealing, and a load concentrator surrounding a reservoir-facing port can be used to improve cover-to-reservoir pressure sealing.

In some embodiments, the pressure cover comprises a filter. For example, the pressure cover can comprise a membrane filter, a sintered filter, a fiber-based filter, and/or a track-etched filter. In some embodiments, the filter is positioned within or adjacent one of the through-hole ports and/or openings thereof. In some embodiments, the filter is positioned within or adjacent one of the channels included in the cover and/or openings of the channels.

In some embodiments, the filter is selected to improve the sterility of the reservoir and/or to block particles, contaminants or microorganisms. In some embodiments, the filter is characterized by an effective pore size of less than or equal to 0.4 um, from 0.2 um to 2 um, from 1 um to 10 um, from 5 um to 20 um, from 10 um to 50 um. It is known in the art that filters characterized by an effective pore size of less than or equal to 0.4 um are preferred for maintaining sterility. However, filters, such as Porex 4901 having an effective pore size of 25 um, have been found to be effective for maintaining sterility.

In some embodiments, the pressure cover comprises one or more gaskets. In some embodiments, the one or more gaskets are adapted to permit or improve pressure sealing (although may not be a complete seal). In some embodiments, at least one gasket is disposed on a reservoir-contacting surface of the cover. In some embodiments, at least one gasket is disposed on an equipment-contacting surface of the cover. In some embodiments, the gasket is adapted to permit or improve pressure sealing with a plurality of reservoirs. In some embodiments, the gasket is adapted to permit or improve pressure sealing at a plurality of equipment-facing ports. In some embodiments, the one or more gaskets comprise an elastomer, a flexible material, an o-ring, and/or combinations thereof. In some embodiments, the one or more gaskets are formed by extrusion, casting, injection molding (e.g., reaction injection molding), die cutting, and/or combinations thereof. In some embodiments, at least one gasket is mechanically coupled to the cover by bonding (e.g., using an adhesive tape), clamping, screwing down, bonding, heat staking, welding (e.g., ultrasonically, by a laser), fusion (e.g., solvent-assisted bonding), and/or combinations thereof.

For example, one embodiment of the present invention of the cover includes a port (5) that allows pneumatic (e.g., vacuum) control of an (optionally) chip to communicate through the cover (see FIGS. 2a-2e ). It is not intended that the cover be limited to pneumatic pressure communication only, and it is contemplated that the cover may additionally be in communication with fluidic or electrical connections.

In one embodiment, the cover can include a detector. For example, the cover can include a pressure detector, for example, to measure the pressure occurring on one or more of the reservoirs. Additionally, the cover can include liquid-level sensing to measure the amount of liquid present in the reservoir or to determine whether a particular fill (or depletion) threshold has been passed. This can be done in a variety of ways. In one embodiment, a detection liquid that utilizes optical refractive index differences is contemplated. In this embodiment, air-filled compartments and channels scatter light, while liquid or fluid-filled channels concentrate light. More specifically, the refractive index of the liquid is between 1.3 and 1.5, while the refractive index of air is only 1.0. In one embodiment, each optical detector comprises a pair of IR emitters (SEP8736, 880 nm, Honeywell) and a phototransistor (SDP8436, 880 nm, Honeywell). In this embodiment, IR is chosen over the visible spectrum because it is less sensitive to interference light.

The ability to easily remove fluids from various reservoirs (e.g., to take samples, replenish media, add test agents, etc.) is a desirable feature. A particularly desirable feature is one that allows the use of standard laboratory pipettes and syringes for these operations. However, such fluidic access (particularly using pipettes) requires that the accessed reservoirs be open to the environment. This is particularly undesirable when chips or disposables are transported or used outside of the device, since the openings can provide a means for contamination of the reservoirs. A typical solution to this problem is to include covers that can be applied to one or more of the reservoirs when they are not being accessed. However, simply including covers can complicate the use of the technology, since typically the user must actively install and remove the covers, as well as maintain the covers in a sterile manner near the device.

One solution is to include means for automatically removing and/or installing the cover as part of the system (whether integrated into the culture equipment or integrated into a separate module). For example, the system may include a mechanical actuator that engages a cover installed on a deployed perfusion disposable and can remove it before engaging the pressure system. The mechanical actuator can reinstall the cover upon removal of the perfusion disposable. In an alternative embodiment, the system includes means for applying a cover to the perfusion disposable prior to or upon removal of the cover, e.g., a cover originating from a magazine of stored covers.

A disadvantage of systems having means for automatically removing and/or installing the cover (as discussed in the previous paragraph) is that they require one or more mechanical actuators, which can make the task difficult in practice. Another challenge is: the design of the reservoir and in particular its opening is aimed at satisfying the requirements for liquid access (manual sampling or refilling using a pipette), pressure-actuated systems (e.g. ensuring a good pressure seal to the device) and manufacturing (e.g. injection moulding of the reservoir). In practice, these requirements may conflict with one another. For example, manual access may require a wide reservoir opening, whereas in contrast a narrower pressure connection may be desirable in order to reduce the forces on the device.

A more preferred solution disclosed herein comprises a "pressure cover" (see FIGS. 2a, 2b, 2c and 2d). The pressure cover can be installed on the reservoir to reduce the possibility of contamination and is designed to remain substantially in place while the perfusion disposable is engaged with the device. To remain substantially in place while engaged with the device, the cover preferably comprises a) one or more features designed to interface with the device (e.g., to accommodate positive or negative pressure), b) one or more features designed to interface with one or more reservoirs (e.g., to create a pressure seal or to minimize gas leakage so that pressure is applied to the reservoir), and c) means for pressure communicating with at least a portion of feature (a) and at least a portion of feature (b). The pressure cover or a portion thereof can be transparent or translucent. This can, for example, allow viewing of the liquid level within the reservoir. The pressure cover can include markings indicating the nature or designation of each reservoir.

In one embodiment of the pressure cover, the opening in the pressure cover (e.g., at the top thereof) can be smaller than the reservoir to reduce the surface area exposed to contaminants and/or to reduce the area applied to the pressure seal. In another embodiment, the cover can include a filter or multiple filters (38) to prevent the ingress of solids and particles (see FIG. 2a ). For example, the cover can include a 0.2 um or 0.4 um filter, which are known to reduce the ingress of bacteria and other contaminants. A variety of materials and technologies can be used for such filters. Since the filter needs to transmit only pressure and not liquid, for example, track-etched filters (e.g., PTFE, polycarbonate, PET), paper filters, porous and expanded materials (e.g., cellulose and derivatives, polypropylene, etc.), and sintered materials (e.g., Porex filters) can be used.

In one embodiment, the cover may include a means that allows gas flow but allows little liquid flow. Examples include hydrophobic porous membranes or filters, gas permeable membranes or filters, and the like. This approach may also help reduce the potential for leakage.

In one embodiment, the cover can include a deformable portion that can be deformed to transmit pressure. For example, this can be an elastic or plastic membrane that extends into the reservoir as positive pressure is applied. Similarly, the cover can include a plunger that is used to transmit pressure from the device to one or more reservoirs. Since the membrane or plunger can apply a back force, care must be taken to ensure that the desired pressure is applied to the interior of the reservoir. This can be done, for example, by a) ensuring that the back force is small or is understood through the design of the membrane, plunger, or operating pressure range, b) measuring the pressure inside the reservoir and using this to control the applied pressure, and c) monitoring the flow generated to control the applied pressure. The deformable portion provides one method of communicating pressure.

One side of the pressure cover (the equipment-facing or perfusion disposable-facing) as well as each opposing surface (the equipment and perfusion disposable features interacting with the pressure cover) can be designed to allow for pressure sealing in a number of different ways. In one embodiment, the invention contemplates one or more regions comprising one or more resilient or flexible materials. In one embodiment, this is accomplished by one or more gaskets (see FIG. 2a ), which may be made of, for example, an elastomeric or flexible material (e.g., silicone, SEBS, polypropylene, Viton, rubber, etc.). The gaskets may be formed in a variety of ways, such as by flat sheet cutting, by o-rings (not necessarily round or cross-sectional), etc. In one embodiment, this is accomplished by one or more raised portions that act as load concentrators (see FIG. 2c ). Without wishing to be bound by theory, these act to localize the sealing force, thereby creating an increased localized sealing pressure. These raised portions potentially engage a gasket or flexible material on the opposing surfaces. Care should be taken to design the shape of the raised portions (particularly the portions of the shape that engage the opposing surfaces), as this shape can have a substantial effect on the required sealing pressure. Various shapes are contemplated (e.g., rectangular, triangular, trapezoidal, semicircular or circular sections, etc.). In one embodiment, the sealing teeth have a trapezoidal shape for improved sealing (see FIG. 2c ). Alternatively, the gasket can be incorporated into the reservoir or lid in the form of an overmolded elastomer (e.g., silicone, SEBS, etc.). The overmolded elastomer itself then has a suitable shape (e.g., a tooth or o-ring semicircular section) to act as a seal.

The approach need not be limited to a single design. In one embodiment, the invention contemplates a combination of one or more regions comprising one or more elastic or flexible materials. Furthermore, the gaskets or ridges may be implemented along the reservoirs, isolated from each other in terms of applied pressure, or may comprise two or more reservoirs, which may reduce complexity. In one embodiment (see FIG. 2d ), the passages surround all chambers of the reservoir chamber-cover assembly seal, isolating each chamber from the other. In one embodiment (see FIG. 2d ), there are two reservoirs, each having an injection chamber (6a, 6b) and an exhaust chamber (7a, 7b), and a separate (optional) vacuum chamber (8) capable of delivering vacuum to the chip or other microfluidic device. In one embodiment (see FIG. 2e ), the reservoir chamber-cover assembly seal comprises sealing teeth (9).

It is not intended that the present invention be limited to designs having a complete pressure seal, as a complete pressure seal may not be required. Rather, some amount of gas leakage may be tolerated, as the equipment can actively control pressure to compensate for leakage. The requirement to obtain a complete seal on one or both sides may be relaxed, simplifying the design and reducing cost.

The pressure cover may be secured or positioned on the reservoir (whether on the perfusion disposable or directly on the chip) in a variety of different ways. Embodiments may include where the liquid or gaseous seal between the cover and the reservoir(s) is even external to the equipment (e.g., the cover may be held in place by something other than the equipment), and where the seal is created by the action of the equipment (e.g., the equipment presses the cover against the reservoir during perfusion). In another embodiment, the invention contemplates a combined approach, whereby, for example, as in the first option above, the cover is designed to create at least a partial seal, but where the seal is confirmed or guaranteed by the action of the equipment, as in the second option above. An advantage of approaches that provide at least some degree of cover sealing to reservoirs that are external to the equipment is that they may reduce the risk of spillage and contamination (e.g., due to handling or transport).

Examples of approaches to securing or placing the pressure lid include (regardless of which of the three approaches they fall into): a) the lid can simply be placed over the reservoir or perfusion disposable (which may be assisted by an overhand portion of the lid, so that the lid cannot simply be slid off); b) the lid can be screwed, glued or pinned in place; and c) the lid can be clipped in place. In an alternative embodiment, it can also be held down by a spring, e.g. a hinged lid has a spring that applies a force to close the lid.

The clip feature may be present in the cover, the perfusion disposable, the chip, or a combination thereof. Additionally, some embodiments utilize a separate substrate that provides a clipping member (i.e., a separate piece for clipping the cover in place). An advantage of the clipping approach is that it can facilitate easy application and removal of the cover while still securing the cover in place. The clipping may be optional, for example, it may be applied when shipping or transporting the device, and may be ignored during regular use.

In some embodiments, the cover is asymmetrical or includes a lock-and-key feature to ensure that the cover is properly oriented relative to the perfusion disposable and/or equipment.

Several features of a perfusion disposable (PD) device could potentially be incorporated into the "chip" itself or into different devices coupled to the chip. If the reservoir is incorporated into the chip, for example, a pressure cover directly over the chip could be used.

Although the pressure shroud has been discussed above in connection with pressurizing one or more reservoirs within a perfusion disposable or a perfusion manifold assembly, it is not intended that the pressure shroud be limited to use with these embodiments. Indeed, it is contemplated that the pressure shroud may be used with other microfluidic devices. The pressure shroud may be movable or removably attached to the other microfluidic device to allow improved access to the member (e.g., the reservoir) from within. The pressure shroud may be removed from such other devices, and the other devices may be used without the shroud. In one embodiment, the other microfluidic device comprises cells on the membrane and/or within or on one or more microchannels.

B. Tray System

It is desirable to be able to remove chips and/or perfusion disposables from the device without having to remove the device itself, for example, from the incubation area. It is also desirable to be able to remove groups of chips and/or perfusion disposables together. This is because operations performed on the chip/disposable (e.g., media replenishment, agent administration, sample collection) often have to be performed in batches at one time, regardless of whether the operations are performed automatically or manually. It is only convenient to remove groups of chips/disposables at one time if this aids in transport, for example, to a biosafety cabinet or culture hood.

To address this need, in one embodiment, the present invention contemplates a system in which perfusion disposables can be inserted into or removed from a device (or module) in groups by a tray system (see FIG. 6 ). For example, this embodiment allows each device to accommodate two trays (or racks) each having six perfusion disposables (8a and 8b).

In one embodiment, the tray (or rack) (32) can facilitate alignment of the perfusion disposable (10) with the equipment (30) (e.g., alignment of the reservoir or port locations with corresponding pressure or fluid connections included in the equipment). This can be accomplished in a number of ways, such as by providing positioning features within the tray for the perfusion disposable (or any additional member that holds them) and by providing positioning features within the equipment for the tray and alignment features (57) for the perfusion disposable (see FIG. 10b ). Features that may be used to facilitate such alignment include reference surfaces, pins, guides, shaped surfaces (e.g., fillets and/or chamfers), springs or resilient members, and the like, to facilitate alignment. These may be incorporated into the tray, the equipment, the perfusion disposable, or a combination thereof.

The tray may optionally be designed to capture leaks originating from the perfusion disposable or from the equipment connection. The tray may optionally include one or more optical windows that facilitate microscopic observation or inspection. This allows the tray to be installed in a microscope or other inspection device, thereby allowing the chips to be observed without having to remove each disposable from the tray. Correspondingly, the tray may optionally be designed to minimize the imaging working distance, e.g., to lie flat on or fit within a microscope stage. The system may optionally include means for retaining one or more perfusion disposables within the tray. For example, the perfusion disposable may be clipped to the tray, and the clip feature may be present on the perfusion disposable, the tray, an additional substrate, or a combination thereof.

In some embodiments, the tray system includes one or more sub-trays (or nests) (47) that fit into the carrier tray (32) (see FIG. 8a ). The sub-trays allow for the simultaneous removal of a subset (e.g., three) of perfusion disposables from the tray. This may be useful, for example, when one or more operations performed on the chip/disposable benefit from a small number of chips present in the carrier tray. For example, in some instances, the inventors prefer to place no more than three disposables on the microscope stage at a time, thereby minimizing the time the chips/disposables spend outside of their desired incubation and perfusion environment. Consequently, this embodiment includes a carrier tray (32) supporting two sub-trays (47), each of which supports three perfusion disposables (10) (see FIG. 8a ).

The sub-tray can facilitate alignment of the perfusion disposable with respect to the device. This can be accomplished in a number of ways, such as by providing positioning features for the perfusion disposable within the tray, by providing positioning features for the sub-tray within the carrier tray, and by providing positioning features for the carrier tray within the device. Features that may be used to facilitate such alignment include reference surfaces, pins, guides, shaped surfaces (e.g., fillets and/or chamfers), springs or resilient members, and the like, to facilitate alignment. These may be incorporated into the carrier tray, the sub-tray, the device, the perfusion disposable, or a combination thereof. For example, the present invention contemplates embodiments in which the perfusion disposable is aligned to the sub-tray, the sub-tray is aligned to the carrier tray, and the carrier tray is aligned to the device (see FIGS. 9A and 9B ). It is not intended that all of these alignments be required, and indeed some steps in the alignment may be omitted. For example, any of the features described above could be used to align the sub-tray directly to the equipment, without requiring a carrier tray for alignment purposes.

The sub-tray may optionally be designed to capture leaks originating from the perfusion disposable or the equipment connection. The sub-tray may optionally include one or more optical windows that facilitate microscopic observation or inspection. This allows the sub-tray to be installed in a microscope or other inspection device, thereby allowing the chips to be observed without having to remove each disposable from the tray. Correspondingly, the sub-tray may optionally be designed to minimize the imaging working distance, for example, by lying flat on or fitting within a microscope stage. The system may optionally include means for retaining one or more perfusion disposables within the sub-tray. For example, the perfusion disposable may be clipped to the sub-tray, and the clip feature may be present on the perfusion disposable, the sub-tray, an additional substrate, or a combination thereof. The system may optionally include means for retaining the sub-tray within a carrier tray. For example, a sub-tray may be clipped to a carrier tray, with the clip feature being on the sub-tray, the carrier tray, an additional substrate, or a combination thereof.

It may be convenient to partition some desired features between the carrier tray and one or more sub-trays. For example, the sub-tray may provide an optical window, and the carrier tray may be designed to trap leakage. For example, it may be desirable to include a sub-tray even if the carrier tray is designed to support only one sub-tray.

The same equipment may support different tray or sub-tray types, as well as different numbers of trays. For example, the equipment may accommodate two different tray types, each of which may be designed for a different type of perfusion disposable. In such cases, the trays may essentially act as adapters to adapt different perfusion disposable types to the same equipment.

In one embodiment, the invention also contemplates a microscope stage, stage insert or adapter (e.g., plunging stage insert) designed to receive one or more chips, perfusion disposables, trays or sub-trays. These can facilitate "loading" a number of chips for imaging while the chips remain securely on the stage (thereby avoiding, for example, shaking as the microscope stage moves).

C. Interlocking of perfusion disposables and equipment

In one embodiment, the present invention contemplates a pressure-driven system for biological culture in a fluidic device that applies pressure (whether positive or negative) to one or more fluidic elements. These fluidic elements include, for example, chips, reservoirs, perfusion disposables, pressure sheaths, or combinations thereof. In such a system, an equipment connection interfaces with each fluidic element or elements for applying pressure as needed. Such connection typically involves establishing a gas seal, although in some embodiments a tight seal is not required (e.g., pressure regulation can maintain a desired pressure despite gas leakage). Without loss of generality, the following description illustrates establishing a seal, but is also intended to include embodiments in which no seal is required.

In the present disclosure, systems and methods are contemplated for establishing a pressure connection between a biological culture device and one or more fluidic elements. In particular, in one embodiment, a system is contemplated wherein one or more fluidic elements are raised to contact one or more pressure manifolds included in the device, or wherein the one or more pressure manifolds are lowered to contact the one or more fluidic elements, or a combination thereof. In some embodiments, the raising or lowering simultaneously engages multiple fluidic elements and the device (e.g., via a single actuation or single movement) (see FIGS. 9A and 9B ), and simultaneously connects multiple microfluidic devices (e.g., one or more embodiments of a perfusion manifold assembly discussed herein).

Some embodiments in which the fluid element is raised include one or more platforms having one or more fluid elements disposed thereon. In such embodiments, the one or more platforms can be raised to effect said raising of the one or more fluid elements (FIG. 6). In some embodiments, the equipment or system includes mechanical means (35) to manually effect said raising or lowering in relation to said establishment of the pressure connection. Such mechanical means (35) for manual actuation can include movement of a user-accessible control surface, which can include, for example, a level, a pull/push knob, a rotary control, or a combination thereof.

In some embodiments, the equipment or system includes a mechanical actuator (51) to facilitate the raising or lowering associated with said establishment of the pressure connection (see FIGS. 9A and 9B ). Such mechanical actuators include, for example, one or more pneumatic components (52) (e.g., a cylinder), hydraulic components (e.g., a cylinder), a solenoid, an electric motor, a magnet (e.g., a fixed magnet that is mechanically moved into place), or a combination thereof. In some embodiments, the mechanical actuation can be computer controlled. In some embodiments, the mechanical actuation is augmented by manual control (e.g., using any of the means for mechanical control described above), for example, to provide manual retraction. A user interface on the equipment can control this process.

Regardless of whether the operation is manual or automated, in some embodiments, the system further comprises one or more mechanisms for increasing the applied mechanical force. This may be desirable to provide sufficient force on the pressure connection to obtain a sufficient or sufficiently firm seal. Such mechanisms for increasing the applied mechanical force may include levers, cams, pneumatic or hydraulic amplifiers, or combinations thereof.

In some embodiments, the mechanical motion can be controlled and/or restrained using various mechanical components or designs known in the art. These mechanical components or designs include, for example, rails, guide rods, pivots, cams, four-rod linkages, etc. It is important to note that the rising or falling motion can be, but need not be, linear. For example, in some embodiments, a rotational motion (e.g., in the case of a pivot) or a composite motion (e.g., in the case of a linkage) is desirable.

While the description has described the raised or lowered features as being on the upper side of the bottom of the various substrates, those skilled in the art will appreciate that the description can also apply to lateral movement or movement along other axes (not necessarily linear movement), and can apply to features present on any side or orientation. Additionally, while the description above suggests that one or more fluid elements are disposed below one or more pressure manifolds, those skilled in the art will appreciate that the pressure manifolds can instead be below the fluid elements (e.g., the pressure connections can be disposed on the lower surface of the perfusion disposable).

This embodiment (illustrated in the attached drawings) includes two mechanics, each of which allows six perfusion disposables to be connected to the pressure manifold (50) in a single motion. In this embodiment, the pressure manifold is lowered into contact with the perfusion disposable (or optionally into contact with a pressure cover covering the perfusion disposable) using an electrically controlled pneumatic actuator (FIG. 9b). The force of the actuator is directed using a cam system, which also increases the applied force due to its mechanical advantage. The illustrated mechanism is also bi-stable, i.e., when the actuator pushes the manifold up or down, the force can be released while maintaining the position of the manifold. This may aid in heat reduction.

D. Pressure Manifold and Distribution Manifold

In many applications of pressure-driven systems, it is desirable to distribute one or more pressure sources to two or more fluid elements (e.g., fluid chips, perfusion disposables, reservoirs, pressure covers, or combinations thereof). For example, it may be desirable for two or more perfusion disposables to share a single set of pressure regulators to reduce the number of regulators in the system (as opposed to providing a different set of regulators for each perfusion disposable, for example).

In one aspect of the present disclosure, the device comprises one or more distribution manifolds. The distribution manifold comprises one or more fluid conduits (e.g., gas channels or tubes) adapted to distribute one or more pressure sources to two or more fluid elements (e.g., fluid chips, perfusion disposables, reservoirs, pressure sheaths, or combinations thereof). Correspondingly, the distribution manifold can comprise one or more pressure inlet ports, which can be adapted to communicate with one or more pressure regulators, for example (each inlet port can communicate with a single or multiple regulators). In one embodiment, the distribution manifold can also have pressure regulating components (valves, pressure sensors, pressure sources) integrated into the manifold itself. Similarly, the distribution manifold can comprise two or more connections, which can be adapted to communicate with one or more fluid elements, for example. In some embodiments, the two or more connections comprise at least one region comprising an elastomeric or flexible material. Examples include gaskets, o-rings, etc. made of materials including silicone, SEBS, polypropylene, rubber, Viton, etc. Such regions including elastomeric or flexible regions can help provide or improve fluid sealing. Such elastomeric or flexible regions can also be included in a pressure manifold rather than a distribution manifold to provide similar benefits.

In addition to distributing pressure, which may be used, for example, to provide pressure-driven flow, the distribution manifold may distribute pressure used to generate gas flow within the fluidic member for other purposes, such as to provide mechanical compression or compression (e.g., to actuate mechanical force in an organ on a chip). Furthermore, the distribution manifold may optionally dispense one or more liquids. Such liquids may include, for example, a cleaning solution, a disinfecting solution, a working liquid (e.g., for liquid-handling or flow control purposes), tissue-culture media, a test agent or compound, a biological sample (e.g., blood), or combinations thereof. In some embodiments, the distribution manifold may include a working fluid, a membrane, and/or a plunger arranged to transmit the pressure. For example, the use of a working fluid may reduce the amount of gas required to establish a desired pressure, or may facilitate more precise volumetric control. The membrane, plunger, and/or working fluid can be used to isolate the fluids used in different components of the distribution manifold (e.g., isolating the 5% CO2 tissue-culture gas on the "reservoir side" of the distribution manifold from the dry air on the working side).

In many applications, it is desirable to allow proper functioning of the equipment even when fewer fluid elements are engaged than the equipment can accommodate. For example, it is often desirable to support proper operation of the equipment, even when a distribution manifold is designed to interface with six perfusion disposables, when only four perfusion disposables are present. For example, it may be undesirable to have gas escape through the intended connection to a removed perfusion disposable, and thus the gas escape could reduce the gas pressure or deplete the gas supply. These considerations are relevant even in the absence of a distribution manifold (i.e., in the presence of a non-distributed pressure manifold).

According to one aspect of the present disclosure, a pressure manifold (or more specifically, a distribution manifold) can include one or more valves adapted to controllably close one or more fluid (e.g., gas) conduits contained within the manifold. A variety of suitable valves are known in the art, such as pinch valves, screw valves, needle valves, ball valves, spring-loaded valves, poppet valves, umbrella valves, Belleville valves, and the like. In some embodiments, the one or more valves are user controlled. For example, the user can configure the valves to match the configuration of the perfusion disposable being used. In some embodiments, the one or more valves are electronically controlled. For example, software can configure the valves based on knowledge of the experimental setup or other information available to it. In some embodiments, the one or more valves are controlled by sensing whether an intended fluid absence is present, for example, to close a gas line when the fluid absence is removed. Such sensing may include electrical means (e.g., contact switches, circuit breaker conductors), optical means (e.g., optical gates), magnetic means (e.g., magnetic switches), or mechanical means (e.g., levers, buttons). In some embodiments, the one or more sensing elements influence the one or more valves using embedded software or electronic hardware. In some embodiments, the one or more sensing elements directly influence the one or more valves (e.g., by mechanical coupling with the valve or by electrical signaling to the valve). As a specific example, the presence of the perfusion disposable may act to depress a protruding feature, which in turn affects the state of the valve. In some embodiments, such a configuration is well suited for pinch valves, spring valves, poppet valves, or umbrella valves, since the depressed protruding feature can act directly on the valve to increase flow rate.

In some embodiments, it is desirable or convenient to include one or more of the valves at one or more of the connections with the fluid element. This may be desirable, for example, because many successful valve designs are known that respond to forces present at the outlet. Examples of such valves include Schrader valves, Dunlop valves, Presta valves, umbrella valves, variations thereof, and related valves. As a specific example, a Schrader valve may be incorporated at the connection to the pressure cover, such that when the pressure cover is present, it acts to press against the central stem of the Schrader valve, thereby allowing gas flow.

As described above, valves suitable for inclusion in a connection to a fluid member often have their control feature located centrally on the valve (e.g., the pin of a Schrader valve) ( FIG. 11a ). However, this can be difficult in some potential embodiments, since a corresponding feature must be provided on the fluid member to depress this central control feature. An alternative approach is described herein. As shown in FIGS. 11a and 11d , a pressure manifold (50) or a distribution manifold may include a valve (59), such as a Schrader valve (or any of the valves described above), and may further include a shuttle (61). The shuttle includes a first surface facing a potential fluid member location, and a second surface facing the valve. The first surface is designed to accept contact from the fluid member at a desired location. For example, the first surface may be designed to receive contact from the periphery of a port, such as may be present on the pressure cover (11) ( FIG. 11d ). The second surface is designed to mechanically engage with a control surface of the valve, which may be, for example, at the center of the valve. An additional advantage of this approach is that the thickness of the shuttle can be adjusted to control, for example, the distance from the fluid body that the valve will open.

As further illustrated in FIGS. 11A and 11C , the connection portion may optionally be at least partially covered by an elastic, flexible or deformable substrate, such as a flexible membrane (e.g., a silicone membrane) (60). The presence of such an elastic, flexible or deformable substrate may assist in sealing the fluid member to the manifold (50). The elastic, flexible or deformable substrate may be, for example, a membrane, a gasket or a suitably shaped plug, which may comprise, for example, silicone, SEBS, Viton, polypropylene, rubber, PTFE, or the like. As illustrated, the elastic, flexible or deformable substrate may be held in place by capturing it by an additional component (e.g., a cover plate (63) in this embodiment). However, the elastic, flexible or deformable substrate may also be held in place in various other ways, such as by bonding, adhering, welding, etc.

Hereinafter, the desired functionality of the embodiments illustrated in FIGS. 9b, 11a and 11d will be exemplified: a pressure cover (11) of a flow manifold assembly (10) having a raised portion around an equipment connection is brought into contact with the pressure manifold (50). As the cover is advanced closer to the valve, the raised portion of the cover begins to form a pressure seal against the silicone membrane of the manifold. As the cover advances, the shuttle slowly moves upward and at some point begins to press against the center pin or poppet (65) of the Schrader valve (59). However, according to the example, the shuttle will be designed such that a sufficiently good gas seal is formed before the pin of the valve is sufficiently pressed to open the Schrader valve (59). Once the valve is opened (and ideally not before), gas can flow between the manifold (50) and the pressure cover (11). It is important to note that in this embodiment, the Schrader valve does not sense the presence of the perfusion disposable (or pressure cover) as a single unit, but rather independently senses the presence of each pressure-cover ridge. Such embodiments may provide an additional advantage in that they can accommodate different configurations of pressure covers or perfusion disposables, for example, a configuration that uses only four of the five ports shown.

FIG. 10a illustrates one embodiment of a PD mating surface (54) of a pressure manifold (50), which is a distribution manifold, and illustrates elastomeric regions that act as gaskets to improve the gas seal against the fluidic voids. In this embodiment, the gas seal can be formed by compressing these elastomeric regions against a raised portion present on top of a pressure cover (11) disposed on a perfusion disposable (10). The illustrated distribution manifold (50) can distribute pressure (positive or negative) used to induce pressure-driven flow to each of the six pressure covers, as well as pressure (positive or negative) used to actuate mechanical stretch within the long-term device on the chip contained therein (in this example, each of which is disposed within a perfusion disposable, said perfusion disposable being covered by a pressure cover). The illustrated distribution manifold includes several Schrader-like valves (see FIG. 11d ).

As the manifold engages the PD, the valve seal engages the sealing teeth or ridges (see FIG. 2c) on the top of the cover to form a seal for transferring pressurized gas from the manifold to the storage chamber. The poppet (65) (FIG. 11d) acts as a backing to provide a hard surface against the sealing teeth on the cover to compress the valve seal. This transfers the load from the cover to the Schrader valve (59), thereby operating it when the PD is in position. At the same time, the Schrader valve (or similar type of valve system) engages the PD cover, thereby operating all gas from the pressure regulator to the PD. When the PD is not in its respective position, the valve prevents any gas flow.

A spring shuttle (55) (Fig. 10b) provides a load to the cover assembly (11) to create a reservoir chamber-to-cover assembly seal (e.g., a pressure cover-to-reservoir seal) (Fig. 2d). In operation, when the PD is engaged, there is deflection of the valve seal and displacement of the poppet (65).

Alternatively, a cover compressor (Fig. 10c) provides a load to the cover assembly to create a reservoir chamber-to-cover assembly seal (e.g., a pressure cover-to-reservoir seal).

In one embodiment, each valve assembly has an optional spring, flexure or elastic component built in that allows pressure to be applied independently to each seal. In one embodiment, the spring (or similar member) is an integral part of the valve function, but may have additional functionality by being used to apply pressure to the sealing teeth on the reservoir cover. The spring (or similar member) may be operative to restore the shuttle and apply pressure to the fluid element, thereby providing or improving a gas seal. By applying this load independently to each sealing element on the cover, a more robust design is created with respect to both variations due to manufacturing tolerances and how multiple PDs are loaded onto the equipment.

In some embodiments, one or more of the described valves are software or user controlled. For example, the user or software may wish to block gas flow even when a fluid member (e.g., a perfusion disposable) is present at the corresponding connection. For example, this may be desirable if the user suspects or the software or a sensor detects that the fluid member is damaged and is causing excessive gas flow into the fluid member. The pressure manifold (whether a distribution manifold or not) may additionally include sensors, such as pressure sensors, flow sensors, etc.

E. Pressure and flow control

In one embodiment, a flow rate of from 5 to 200 uL/hr, and more preferably from 10 to 60 uL/hr, is preferred through one or more microchannels of the device. In one embodiment, this flow rate is controlled by the gas pressure applied from the pressure manifold (described above). For example, when 0.5 to 1 kPa is applied, this nominal pressure produces a flow rate of from 15 uL/hr to 30 uL/hr in one embodiment.

In addition to maintaining control over the gas pressure (and thereby control over the flow rate) over time, in some embodiments, the gas pressure that may be applied by the process of engaging or disengaging the manifold to the perfusion disposable must also be addressed. That is, in particular embodiments, the engaging step of the manifold has been observed to produce a pressure spike of as much as 100 kPa for the gas present within the reservoir contained within the perfusion disposable. This may result in a spike in flow rate and/or undesirable pressure on the coupled microfluidic device. In particular cases where the coupled microfluidic device comprises a membrane, the undesirable pressure spike may deform the membrane, result in transmembrane flow, and/or damage any contained cells.

Without being bound by theory, the pressure surge described above may occur because mechanical force applied to the pressure cover by the manifold deforms one or more compliant materials contained in the pressure cover or the perfusion disposable (e.g., compressing any gaskets, etc.). This deformation may act to decrease the volume of gas present in the reservoir, thereby increasing its pressure. The opposite effect of inducing a negative pressure surge may occur during manifold separation; those skilled in the art will appreciate that while this discussion primarily considers positive pressure surges typical of manifold engagement, it may similarly apply to negative pressure surges that may be typical of manifold separation. Whether positive or negative, surges can be particularly problematic when the gas volume in the reservoir is small (which may occur when the liquid volume in the reservoir is large, e.g., greater than 3 milliliters, and particularly greater than 5 milliliters in preferred embodiments). These engagement surges can take time to dissipate, typically because the excess pressure must be vented. In embodiments where the pressure cover comprises a strainer, the strainer may provide a dominant resistance to exhaust, which dictates the dynamics of pressure surge dissipation. In one embodiment, the present invention contemplates reducing the exhaust resistance in the system to avoid such surges, reduce the size of the surges, and/or reduce the duration of the surges. In one embodiment, the present invention contemplates selecting a strainer to mitigate pressure surges during cartridge insertion and removal.

In this regard, see FIG. 2. FIG. 2a is an exploded view of one embodiment of a cover assembly (11) including a cover or lid having a plurality of ports (e.g., through-hole ports) associated with corresponding apertures (39) in a strainer (38) and a gasket. FIG. 2b illustrates the same embodiment of a cover assembly with the strainer (38) and gasket positioned within (and beneath) the cover. In one embodiment, the strainer for the discharge pressure port is selected for low gas-flow resistance. For example, some embodiments use a 25 micron strainer instead of the 0.2 micron strainer (used at the inlet pressure port) to reduce resistance and to rapidly dissipate manifold-engagement-related gas pressure (discussed above) to avoid prolonged spikes in flow rate. In a special embodiment, filters having an average pore size of 25 um (commercially available from Porex, Filter 4901) do not compromise sterility when the thickness is 1/8 inch. These filters maintain sterility despite their large pore size (much larger than typical bacteria/spores) by creating complex passageways through their thickness, which is significantly thicker than previously mentioned filter membranes/sheets.

It is important to note that the design of the inlet and outlet pressure ports may require different handling with respect to exhaust resistance. For example, in embodiments where the perfusion disposable or microfluidic device includes a resistor, the pressure applied to the resistor side (whether the resistor is installed upstream or downstream of the region of interest) typically does not act directly on the region of interest (which may, for example, include cells). This may be the case, for example, where liquid flow through the resistor creates a pressure drop. Conversely, a pressure surge on the side without the resistor (whether at the inlet or outlet) may act directly on the region of interest, since there may not be sufficient pressure drop to provide some degree of insulation. In the particular example where there is a resistor on the inlet side of the region of interest, a pressure surge on the inlet may create a corresponding surge in flow rate, but the minimal increase in pressure is experienced within the region of interest; conversely, a pressure surge on the outlet may create both a surge in flow rate and a surge in experienced pressure. In some applications, for example where the microfluidic device comprises a membrane, the pressure in the region of interest may be significantly more detrimental than a transient spike in flow rate. Therefore, it may be advisable in this embodiment to include only a low-resistance filter at the discharge port and a more conventional (high-resistance) filter at the inlet port, as these may provide an advantage in flow modulation (as discussed further herein).

Having discussed the interlock/separation surge problem, we now address the problem of controlling gas pressure, particularly in the lower pressure range. Some commercially available pressure regulators (or pressure controllers) advertise an adjustable pressure range with a lower pressure limit that is greater than zero. For example, the SMC ITV-0011 regulator is marketed for pressure control in the range of 1 to 100 kPa (although their linearity has been observed to be poor in the range of 0 to 1 kPa). In some applications, it may nevertheless be desirable to achieve flow rates corresponding to pressures lower than the stated or linear range of the commercially available regulator. Furthermore, the accuracy of commercially available pressure regulators is typically a percentage of "full range," implying that control at the lower pressure limit is characterized by a greater percentage of variability. In some applications, this may translate into poor accuracy or fidelity in pressure control for the lower end of the usable range. In one embodiment, one or both of these challenges are addressed by a form of "pulse-width modulation" incorporated into the pressure actuation method.

In this regard, see FIG. 6 . In one embodiment, the culture module (30) comprises a removable tray (32) for positioning the assembly-chip combination, a pressure surface (33), and a pressure controller (34). In one embodiment, the tray (32) is positioned in the culture module (30) and moved upwardly via the tray mechanism (35) such that the tray (32) engages the pressure surface (33) of the culture module, i.e., the cover or lid (11) of the perfusion manifold assembly engages the pressure surface of the culture module. Rather than the pressure controllers being "on" all the time, they are switched in a pattern of "on" and "off" (or between two or more settings). Thus, the switching pattern can be selected such that the average value of the liquid working pressure in one or more reservoirs corresponds to a desired value. This approach is similar to pulse-width modulation (PWM), pulse-density modulation (PDM), delta-sigma modulation (DSM) techniques and similar techniques known in the electrical engineering arts. In the case of pulse-width modulation, for example, regular switching periods are selected. Within each period, the pressure controller can be switched on for the desired duration at the set pressure or switched off for the remainder of the switching period. The longer the switching-on time is compared to the switching-off time, the higher the overall average pressure applied. The term "duty cycle" describes the ratio of the "on" time to the switching period; a low duty cycle corresponds to a low pressure since the pressure is switched off most of the time. The duty cycle is expressed as a percentage, with 100% being full "on". By using this type of "pulse-width modulation" in combination with a pressure controller, it has been found that it is possible to reliably maintain the average gas pressure to less than 1 kPa using regulators that do not provide linear control in that range. In a special embodiment, the pressure regulator is used in its typical "linear" mode for pressures from 1 kPa to 100 kPa and switched with pulse-width modulation using an "on pressure" of 2 kPa and an "off pressure" of 0 kPa for a mean-pressure setpoint from 0 kPa to 1 kPa. In other examples, pulse-width, pulse-density or delta-sigma modulation can be used to control a mean pressure of 0.3 to 0.8 kPa.

While the disclosed method may involve applying a pulsatile pressure pattern to the pressure cover, it has been experimentally verified that the filter aids in smoothing the pressure exerted on the liquid by the reservoir. Without being bound by theory, the degree of smoothing increases with the resistance of the filter to gas flow and the volume of gas within the reservoir (typically decreasing as more liquid is present). Similarly, the analogy to electrical circuits indicates that shorter switching periods result in greater smoothing. Accordingly, one skilled in the art can select the degree of smoothing by selecting the resistance of the gas filter, setting a lower limit for the gas volume, and selecting the switching period or modulation pattern. It is important to ensure that the pressure regulator controllably modulates the pressure at a rate sufficient to reproduce the designed pressure modulation pattern. In some embodiments, a 0.2 um filter (a porex filter membrane) and a switching period of 10 seconds provide desirable smoothing. In other embodiments, a 0.4 um filter may be used.

Detailed description of preferred embodiments

A. Point-to-point connection

A drop-to-drop connection is contemplated as one embodiment for mounting a microfluidic device in fluid communication with another microfluidic device, such as, but not limited to, mounting a microfluidic device in fluid communication with a perfusion manifold assembly. When the devices are mounted in fluid communication with each other, a bubble (40) can be formed, as illustrated in FIGS. 14A and 14B , wherein a first surface (87) comprising a first fluid port (89) is aligned along a second surface (88) and a second fluid port (90). In one embodiment, the use of a drop-to-drop connection reduces the chance of bubbles being trapped during the connection. Air bubbles are particularly challenging in microfluidic geometries because they are pinned to surfaces and are difficult to flush out by fluid flow alone. They present an additional challenge in cell culture devices because they can damage cells through a variety of means.

In one embodiment, the drop is formed on the surface of the device in the region surrounding and above the fluid path or port as illustrated in FIGS. 15a, 16a, 16b, 16d and 17-21. When the surfaces come close to each other during connection, the drop surfaces are connected without introducing any air bubbles. In practice, it is difficult to maintain alignment and stability of the drop during manual device operation. Additionally, in situations where the coupling number is high, the liquid tends to drain from the device in a rapid and unstable manner. Herein, a number of solutions are described to address both the problem of maintaining a stable drop on the device surface and the problem of guiding the drop-to-drop engagement of two primed devices in a controlled and robust manner.

FIG. 16a illustrates an embodiment of a microfluidic device contacting a fluid source or another microfluidic device, wherein the microfluidic device is approached from the side and the side track is engaged with a portion configured to fit into the side track. FIG. 16b illustrates an embodiment of a microfluidic device contacting a fluid source or another microfluidic device, wherein the microfluidic device is approached from the side and from the bottom and the side track is engaged with a portion configured to fit into the side track, the side track including an initial linear portion and a subsequent angled portion such that when the microfluidic device is engaged and moves with the side track, both lateral and upward movement of the microfluidic device is caused, thereby creating a drop-to-drop connection that establishes fluid communication (FIG. 16c). FIG. 16d illustrates another approach to contacting a microfluidic device with a fluid source or another microfluidic device, where the microfluidic device pivots at a hinge, joint, socket, or other pivot point on the fluid source or other microfluidic device (arrows illustrate the general direction of movement).

FIG. 17 is a schematic diagram showing a confined droplet (22) on a surface (21) of a microfluidic device (16) in a path or port, wherein the droplet covers the entrance to the port and protrudes above the port, and the port is in fluid communication with the microchannel.

FIG. 18 is a schematic diagram showing a confined droplet (22) on a surface (21) of a microfluidic device (16) in the region of a path or port, wherein the droplet rests on a molded pedestal or mount (42), covers the entrance to the port, and protrudes above the port, the port being in fluid communication with the microchannel.

FIG. 19 is a schematic diagram showing a confined droplet (22) on a surface (21) of a microfluidic device (16) in the region of a path or port, wherein the droplet is on a gasket (43), covers the entrance to the port, and protrudes above the port, the port being in fluid communication with the microchannel.

FIG. 20 is a schematic diagram showing a confined droplet (22) in the region of a path or port, where a portion of the droplet is located below a surface (21) of a microfluidic device (16), wherein the droplet is on a shaped recess or depression (44), covers the entrance to the port, and a portion protrudes above the surface, the port being in fluid communication with the microchannel.

FIG. 21 is a schematic diagram showing a confined droplet (22) in the region of a path or port, where a portion of the droplet is located below a surface (21) of a microfluidic device (16), wherein the droplet is within a surrounding gasket, covers the entrance to the port, and a portion protrudes above the gasket.

FIG. 22 is a schematic diagram illustrating a surface modification embodiment using a sticker to confine a droplet on a surface of a microfluidic device (16) in a port, wherein the port is in fluid communication with a microchannel. FIG. 22a illustrates a droplet (22) that spreads from an edge of the sticker and is confined by a surrounding hydrophobic surface. FIG. 22b illustrates a droplet (22) that spreads on a hydrophilic surface of the device and is confined by a surrounding hydrophobic surface (45) created by one or more adhesive layers or stickers on each side of the port, wherein the port is in fluid communication with the microchannel.

FIG. 23 is a schematic diagram illustrating an embodiment of a surface modification utilizing a surface treatment (e.g., chemical vapor deposition, plasma oxidation, Corona, etc. - indicated by the downwardly drawn arrow) in conjunction with a mask (41); in one embodiment, the microfluidic device (16) is fabricated from a naturally hydrophobic material such that upon such surface treatment, the areas without the mask become hydrophilic while the areas with the mask remain hydrophobic. After the surface treatment, the mask can be removed and the channels can be filled with fluid to create droplets that protrude above the surface but are bound by the areas that remain hydrophobic (see FIG. 17 ).

FIG. 24 is a schematic diagram of one embodiment of a drop-to-drop connection scheme that controls droplets using a combination of geometrical features and surface treatments. FIG. 24a depicts an embodiment of a microfluidic device or "chip" including fluidic channels and ports, each having a raised region (e.g., a pedestal or gasket) at each port. When other portions of the device (i.e., portions other than the pedestals or gaskets) are treated (e.g., by plasma treatment) to render them hydrophilic, the naturally hydrophobic pedestals or gaskets can be protected with a mask (illustrated on the pedestals or gaskets as member (41) in FIG. 24a) during the plasma treatment to prevent them from becoming hydrophilic. After the plasma treatment, the mask is removed (e.g., by peeling it away from the surface of the pedestals or gaskets). FIG. 24b illustrates a fluid-filled hydrophilic channel where the drop radii are balanced at each end (i.e., at the port openings), and the drop (22) is confined by a hydrophobic gasket surface. FIG. 24c illustrates a portion of the microfluidic device of FIG. 24b with the upwardly protruding drop (22) approaching (but not yet contacting) a portion of a mating surface of a perfusion manifold assembly having the protruding drop (in this case, the drop (23) protruding downwardly). FIG. 24d illustrates the same portion of the microfluidic device of FIG. 24c with the upwardly protruding drop (22) of the microfluidic device contacting (merging) with the downwardly protruding drop (23) of the perfusion manifold assembly. The drops coalesce in a controlled manner while on the hydrophilic surface but being confined by the hydrophobic surface. As previously mentioned, embodiments are also contemplated in which the microfluidic device (having downwardly protruding droplets) is approached from above the perfusion manifold assembly (having upwardly protruding droplets).

FIG. 25 illustrates an embodiment of a drop-to-drop connected using only a surface treatment (i.e., without a geometry such as a pedestal or gasket). FIG. 25a illustrates an embodiment of a perfusion manifold assembly including fluid channels and ports. When other portions of the naturally hydrophobic mating surfaces (i.e., other than the area surrounding the ports) are treated (e.g., by plasma treatment) to become hydrophilic, the area surrounding the ports is protected by a mask (illustrated in FIG. 25a as a member (41) covering the ports and a small area of the mating surface surrounding the ports) during the plasma treatment to prevent it from becoming hydrophilic. After the plasma treatment, the mask is removed (e.g., peeled away from the mating surface surrounding the ports). FIG. 25b illustrates hydrophilic channels filled with fluid to a predetermined level (e.g., the height of the fluid column). In some embodiments, the formed droplets can resist the pressure exerted by the fluid volume (gravitational head). This is advantageous because it allows for drop-to-drop connection while minimizing the droplet's droplet loading and stabilizing its size. Without being bound by theory, the droplet resists the pressure exerted by the fluid volume because it is balanced by the surface tension of the droplet; this surface tension is determined in part by the droplet radius, which can be controlled using the designs and methods disclosed herein; for example, when the droplet is bound by a hydrophobic region around a port, the radius of its surface is similarly bound.

FIG. 26 is a chart showing (without being bound by theory) the relationship between port diameter (in millimeters) and the maximum hydrostatic head (in millimeters) that a stabilized drop can support, assuming that the fluid has the same surface tension as water (the model does not include a reservoir meniscus). This demonstrates that one can work with a variety of port diameters, selecting one that can support a substantial volume of water column in the channel (typically one that can support substantial back pressure), providing significant process latitude and tolerance for user manipulation. In yet another embodiment, a protruding or protruding drop of a desired size is achieved by adjusting the pressure on the fluid.

The present invention is not intended to be limited to any particular method of controlling droplet size, orientation, or direction. In one embodiment, the present invention contemplates using (or fabricating) a surface engineered to form stable droplets. Such surfaces may be inherently hydrophilic or hydrophobic, or may be treated to be hydrophilic or hydrophobic. The present invention is not intended to be limited to any one technique. However, among the various methods of hydrophilic treatment (e.g., low pressure oxygen plasma treatment, corona treatment, etc.), clean techniques are preferred for treating poly(dimethylsiloxane) (PDMS) microfluidic devices. In one embodiment, the present invention contemplates using atmospheric RF plasma to create a hydrophilic surface (on a generally hydrophobic material). See Hong et al., "Hydrophilic Surface Modification of PDMS Using Atmospheric RF Plasma," Journal of Physics: Conference Series 34 (2006) 656-661 (Institute of Physics Publishing). In one embodiment, a mask (41) is used in conjunction with such plasma treatment, as illustrated in FIG. 23 . For example, the mask can be attached to areas of the surface of the microfluidic device (16) (e.g., made of PDMS or another polymer) prior to the plasma treatment to prevent those areas from becoming hydrophilic (thereby controlling which portions of the PDMS chip become hydrophilic and which portions remain hydrophobic). After the plasma treatment, the mask (41) can be removed ( FIG. 24 ) (typically by simply peeling the mask from the surface). In yet another embodiment, the present invention contemplates utilizing a plasma surface treatment in a fluorinating environment to increase the hydrophobicity of a surface. See Avram et al., "Plasma Surface Modification for Selective Hydrophobic Control," Romanian J. Information Science and Technology , Vol. 11, Number 4, 2008, 409-422.

Alternatively, the surface can have geometric features or configurations that allow the droplet to form or behave in a desired manner. For example, as illustrated in FIG. 18, the mating surface can have a protrusion, platform or pedestal (42) having a geometry that allows the droplet to have a particular dimension. As illustrated in FIG. 19, the surface can also be covered with a structure surrounding the port through which the droplet protrudes, such as a gasket (43) or other mechanical seal, which fills the space between the two mating surfaces (i.e., one surface from the microfluidic device and one surface from the perfusion assembly) to prevent leakage while under compression.

Alternatively (FIG. 20), a portion of the droplet may be positioned in a recess or depression (44) such that a portion of the droplet is below the bonding surface (21) of the microfluidic device, as illustrated in FIGS. 20 and 21. In yet another embodiment, an adhesive patch or sticker (45) may be provided on the surface, as illustrated in FIGS. 22a and 22b, to create a hydrophilic or hydrophobic region on the bonding surface of the microfluidic device.

In yet another embodiment, a combination of geometric features and surface treatments may be applied. For example, a hydrophobic pedestal or gasket may be used (or manufactured) to allow for a smaller droplet size. Most elastomeric polymers used in the manufacture of gaskets are hydrophobic. Such gaskets are commercially available, for example, from Stockwell Elastomerics, Inc. (Philadelphia, Pa.). Meanwhile, M&P Sealing (Orange, Tex.) machines high-quality products, such as soft hydrophobic gaskets, manufactured from materials such as polytetrafluoroethylene ("PTFE"), perfluoroalkoxy ("PFA"), or fluorinated ethylene ("FEP"). These are also contemplated in some embodiments. When other parts of the device (i.e., parts other than the base or gasket) are treated (e.g., by plasma treatment) to make them hydrophilic, the naturally hydrophobic base or gasket can be protected with a mask during the plasma treatment to prevent it from becoming hydrophilic.

In one embodiment, the wall of the port (or at least a portion thereof extending to the mating surface of the microfluidic device) is or becomes hydrophilic. In one embodiment, the wall of the corresponding port (or at least a portion thereof extending to the mating surface of the perfusion assembly) is or becomes hydrophilic. In one embodiment, both the wall of the port of the microfluidic device and the corresponding port (or a portion thereof) of the perfusion assembly are or become hydrophilic.

In one embodiment, the present invention contemplates that the surface is designed to hold a droplet that resists the weight of the liquid in the reservoir (as shown in FIG. 25). This is particularly important in practice, as it allows for a droplet to be easily formed that is placed on the upper device (i.e., when the first device approaches the second device from above). This embodiment allows for a measured amount of liquid to be simply placed on the reservoir (e.g., 100 uL, 75 uL, 50 uL, or some other amount), and the liquid flows into the port to form a droplet and stop on its own. Importantly, this embodiment is not intended to be limited to any particular amount of liquid, and in practice, a precisely measured amount of liquid is not required. In order to form a droplet by this method, it is sufficient to target a particular amount, as long as that amount is below a particular threshold (where the weight of the water overwhelms the surface tension of the droplet and it breaks). This may be somewhat convex, depending on how much liquid is being pushed over it, but the spatial extent of the droplet should be the same.

The present invention is not intended to be limited to a single manner of drop-to-drop connection of microfluidic devices. In one embodiment, as illustrated in FIG. 15A , a first microfluidic device, e.g., an on-chip organ microfluidic device comprising cells that mimic one or more functions of cells in an organ in the body (i.e., mimic one or more functions of cells in an organ in the body, such as cell-cell interactions, cytokine expression, etc.), has droplets that protrude upwardly, while a corresponding droplet on a second microfluidic device protrudes downwardly. In another embodiment, a first microfluidic device, e.g., an on-chip organ microfluidic device comprising cells that mimic cells in an organ in the body or that mimic at least one function of an organ, has droplets that protrude downwardly, while a corresponding droplet on the second microfluidic device protrudes upwardly.

Leaving aside the debate about momentum, gravity alone plays a role in the formation of a stable drop. For example, a chip lying flat on a table does not experience significant forces due to gravity. For example, if the device is tilted as part of the interlocking procedure, the fluid will flow from a higher point to a lower point. Therefore, the orientation of the device, such as having the device face up or down, may be considered as another way to help confine the drop.

An additional aspect of controlling drop volume is the fluidic resistance of the device channels. For example, if the device has small channels, the fluidic resistance may be high enough to maintain a nearly constant drop volume over time, even when there are forces (e.g., gravity or capillary forces) that drive fluid flow out of the device. This is true even at high binding numbers. Adjusting the fluidic resistance may be used as a sole method of "confining a drop" or in combination with other methods, such as controlling the liquid pinning geometry or controlling the wetting properties of the surface; while fluidic resistance may be used to control drop volume, controlling the wetting properties of the surface may help control drop placement.

B. Microfluidic devices

The present invention is not intended to be limited by the nature of the microfluidic device. However, preferred microfluidic devices are described in U.S. Pat. No. 8,647,861 (incorporated herein by reference) and are microfluidic "organ-on-a-chip" devices comprising living cells in microchannels, e.g., cells on a membrane of the microchannels exposed to culture fluid at a predetermined flow rate. The surface and/or membrane of the microchannels can be coated with cell adhesion molecules to support attachment of the cells and promote their organization into tissues. When a membrane is used, the tissues can be formed on the upper surface, the lower surface, or both. In one embodiment, different cells are alive on the upper and lower surfaces, thereby creating one or more tissue-to-tissue junctions separated by the membrane. The membrane can be porous, flexible, elastic, or a combination thereof, and has pores large enough to allow only the exchange of gases and small chemicals, or large enough to allow the movement and passage of living whole cells as well as large proteins. In one embodiment, the membrane selectively expands and contracts in response to pressure or mechanical force, thereby allowing additional physiological stimulation of mechanical forces at living tissue-tissue junctions.

FIG. 33 is a schematic diagram of an exemplary microfluidic device or "organ-on-a-chip" device. The assembled device is schematically depicted in FIG. 33a and includes a plurality of ports. FIG. 33b is an exploded view of the device of FIG. 33a, showing a lower piece (97) having channels (98) in a parallel configuration, and an upper piece (99) having a plurality of ports (2), wherein the tissue-tissue interface stimulation region includes a membrane (101) between the upper piece (99) and the lower piece (97), wherein cell behavior and/or passage of gases, chemicals, molecules, particles, and cells is monitored. In one embodiment, the inlet fluid port and the outlet fluid port are in communication with the first central microchannel, such that fluid can dynamically move from the inlet fluid port to the outlet fluid port through the first central microchannel independently of the second central microchannel. It is also contemplated that fluid passage between the inlet and outlet fluid ports may be shared between the central microchannels. In any embodiment, the characteristics of the fluid flow through the first central microchannel, such as the flow velocity, can be controlled independently of the characteristics of the fluid flow through the second central microchannel, and vice versa.

FIG. 34 is a schematic diagram showing an embodiment having two membranes (101 and 102) with cells (103) inside the device in a first channel, and in contact with fluid channels (104 and 105), with arrows showing the direction of flow. The three channel device allows for tracking the movement or motion of cells, such as lymphocytes, vascular cells, nerve cells, etc. In one embodiment, the membrane (101) is coated on its upper surface with lymphatic endothelium and on its lower surface with stromal cells, and the second porous membrane (102) is also coated on its upper surface with stromal cells and on its lower surface with vascular endothelium. The movement of these vascular and stromal cells can be monitored. Alternatively, a third type of cell can be placed in the center (103) and movement through the membrane can be monitored (e.g., by imaging or by detecting cells in the channel or channel fluid). The membrane can be porous or can have grooves that allow cells to pass through the membrane.

In one embodiment, the three channel device is used to measure the cell behavior of cancer cells. Tumor cells are placed in a central microchannel, for example, surrounded at the top and bottom by a layer of stromal cells on the surface of the upper and lower membranes. A fluid, such as cell culture medium or blood, enters the vascular channel. A fluid, such as cell culture medium or lymph, enters the lymphatic channel. This configuration allows researchers to mimic and study tumor growth and invasion into blood vessels and lymphatics during cancer metastasis. The membrane may be porous or may have grooves that allow cells to pass through the membrane.

C. Seeding the device with cells

In several embodiments described above, the microfluidic chip or other device comprises cells. In some embodiments, the cells are seeded directly onto the chip. However, in other embodiments, the chip is contained within a carrier, and the carrier is mounted on a stand to facilitate cell seeding. FIGS. 35A-C illustrate one embodiment of a "seeding guide" and stand. In one embodiment, the seeding guide is engaged with a carrier containing the microfluidic chip and supports the chip upright (e.g., for upper channel seeding) and upside down (e.g., for lower channel seeding) during various stages of seeding and/or coating (e.g., ECM coating), thereby improving aseptic technique. FIG. 35A illustrates one embodiment of a stand (100) assembled, i.e., with two end caps (106, 107) engaged with side panels (108, 109). FIG. 35b shows a chip (16) and carrier (17) engaged by the seeding guide, with the seeding guide approaching the stand (100). FIG. 35c shows six carriers (17) with chips, each engaged with a seeding guide mounted on the stand (100). The seeding guide is adapted to receive a chip carrier (e.g., in a manner similar to how a skirt engages a chip carrier); and the same chip carrier can be connected to a perfusion manifold assembly (after separation from the seeding guide) after coating and/or seeding. The seeding guide is designed to hold the chip (either upright or upside down) so that the port does not contact the table top or any other surface. This is to avoid contamination of the chip through such contact. Additionally, the seeding guide or holder may facilitate access to the chip via a pipette and/or needle, and optionally assist in insertion into the chip port using the guide features.

In one embodiment, the invention contemplates a method of seeding, comprising the steps of: a) providing a stand having a portion configured to receive i) a chip at least partially contained in a carrier, ii) cells, iii) a seeding guide, and iv) at least one seeding guide in a stable mounting position; b) engaging the seeding guide with the carrier to create an engaged seeding guide; c) mounting the engaged seeding guide on the stand, and d) seeding the cells onto the chip (e.g., using a pipette tip) while the seeding guide (along with the carrier and chip) is in the stable mounting position. In one embodiment, the microfluidic device or chip comprises an upper channel, a lower channel, and a membrane separating at least a portion of the upper and lower channels. In one embodiment, the microfluidic device or chip comprises cells on the membrane and/or within (or on) one or more of the channels following the seeding of step c) (e.g., the upper channel is seeded). In one embodiment of the method, a plurality of seeding guides are mounted on a stand and allow a plurality of chips to be seeded with cells. The guides have a number of functions, including a) keeping the chip surface sterile during handling, b) properly guiding pipette tips into the ports during seeding, c) clearly labeling the channels of the chip (e.g., distinguishing between upper and lower channels), and d) allowing for loading of liquid in the channels onto the chip (as well as loading of cells that have already been seeded or functionalized with ECM onto the chip). The stand also has a number of functions, including a) maintaining the chip level to allow cells to be distributed evenly across the membrane, b) allowing the guides to be flipped over for seeding of the lower channels, and c) allowing the user to hold and store multiple seeded chips at one time. Thus, in one embodiment, after the seeding of step c), the method continues with the step of flipping the chip over to seed the lower channels.

experiment

Example 1

Conditions for bonding a capping layer (Fig. 2, member 13) to a backplane (14) were tested. An extruded SEBS sheet was bonded to a high temperature embossed plate. The SEBS sheet was designed to act as a capping layer for the channels formed in the COP through the high temperature embossing process and as a fluid and gasket for the bonded components. Testing confirmed that 1 mm thick SEBS provided a better fluid seal between the reservoir and the backplane. The high temperature embossed plate was fabricated from Zeonor 1420R. The SEBS materials used were as follows:

A. Thickness: 1mm, Material: Kraton G1643, Mfg Process: Extrusion

B. Thickness: 0.2mm, Material: Kraton G1643 +5% Polypropylene, Mfg Process: Extrusion

The oven process was used in comparison with the laminator. The laminator showed limitations due to not bonding properly. However, the oven process showed the following:

In some embodiments, the fluid layer is sealed with a film. The film can be a polymeric, metallic, biological film, or a combination thereof (e.g., a laminate of multiple materials). Examples of materials include polypropylene, SEBS, COP, PET, PMMA, aluminum, and the like. Specifically, the film can be elastomeric. The film can be secured to the fluid layer by adhesives, thermal lamination, laser welding, clamping, and other methods known in the art. The film can further be used to secure and potentially fluidly connect additional components to the fluid layer. For example, the film can be used to attach one or more reservoirs to the fluid layer. In an exemplary embodiment, the film is a thermal lamination film comprising EVA or EMA. In an exemplary embodiment, the film can be first laminated to the fluid layer using a thermal treatment, and then a second thermal treatment can be used to attach one or more reservoirs to the fluid layer. In a different embodiment, the film comprises SEBS, which is known to be bondable to a variety of materials including polystyrene, COP, polypropylene, and the like, either with the aid of heat treatment or one or more solvents. In this embodiment, the SEBS film can be laminated to the fluid layer (either with the aid of heat treatment or a solvent), and a second treatment can be used to bond one or more reservoirs to the fluid layer. There are several potential advantages to using films that are elastomeric, deformable, or flexible, or that reflow during the bonding process. These advantages include, for example: potentially conforming to the fluid layer or other bonded component (e.g., the reservoir), thereby alleviating manufacturing tolerances (e.g., for flatness or planarity of the manufactured part), potentially simplifying parallelism or alignment required during bonding (e.g., the film can deform to absorb errors in parallelism), and acting as a gasket, thereby creating a fluid seal, for example, between a fluid backplane and a reservoir. SEBS is particularly advantageous as a bonding film because it can be bonded without significant reflow at moderate temperatures (typically below 100C). Reflow can be undesirable as it carries the risk of filling and clogging the fluid channels. Because there is no significant reflow, SEBS can better maintain the dimensions and structure of the fluid channels and other features of the fluid layer than reflowable materials (e.g., typical thermal lamination films). In different embodiments, the film thickness can range from 10 um to 5 mm. The film can include a variety of fluid ports or channels. The film need not be flat and can have a variety of three-dimensional configurations.

Example 2

In this example, one embodiment of a protocol for chip activation is discussed. This example assumes that all work is performed under a hood using aseptic technique and that all work areas are sterile (or made sterile).

Part I: Chip Manufacturing

A. Spray 70% ethanol on the outside of the chip package and wipe it before placing it in the hood.

B. Open the package inside the hood and take out the chips in the chip carrier (take them out together).

C. Place the chip in the chip carrier into a large sterile dish.

i. Handle chip carriers only by their wings. Always use forceps to handle chips. Connect the chip surface to the cell culture area. Avoid contact with the chip surface by hand and hold the chip unit until it is flat.

D. Before opening, allow the vial of Emulate Reagent 1 (ER1) powder (containing cross-linker) to fully equilibrate to ambient temperature to prevent condensation within the storage container - ER1 is sensitive to moisture and light.

E. Turn off the lights in the biosafety hood.

F. Reconstitute the powder with reagent 2.

i. Add 1 ml of emulsion reagent 2 (ER2) (containing buffer) directly to the ER1 storage container and mix thoroughly by inverting three times.

ii. Cover the ER1 solution with tin foil to prevent deterioration due to light.

G. Wash the chip.

i. Orient the chip horizontally within the hood.

ii. Pipette 100 ul of ER2 solution using the tip.

iii. Place the pipette in a completely vertical position and insert it into the lower channel - if the port is difficult to find, feel the surface near the port to explore.

iv. After finding the port, insert the tip into the port (connect it firmly).

v. Flush with 100 ul of ER2 solution, continuing to press the pipette plunger (if you see fluid coming out, the flush was successful; if you see fluid coming out of the same injection port, the tip was not injected properly and repeat step iv).

vi. To remove the tip, gently press the chip body using sterile forceps to remove the tip, while continuing to press the pipette plunger.

vii. Aspirate the exhaust flow.

viii. Repeat the same procedure to clean the upper channel.

ix. After washing, empty the upper channel first by suction, and then empty the lower channel.

H. Inject ER1 solution into both channels.

i. Pipette 30 ul of ER1 solution using the tip.

ii. Use a pipette tip to probe the injection port in the lower channel on the upper surface of the chip near the port.

iii. After finding the port, insert the tip into the port (connect it firmly).

iv. Inject 30 ul of ER1 solution and keep pressing the pipette plunger (if you see the discharged fluid coming out, the injection was successful, if you see the fluid coming out of the same injection port, the tip was not injected properly and repeat step ii).

v. To remove the tip, gently press the chip body using sterile forceps to remove the tip, while continuing to press the pipette plunger.

vi. Aspirate excess fluid from the chip surface (avoiding contact with the ports).

vii. Repeat the same procedure for the upper channel using 50 ul of ER1 solution.

viii. Avoid introducing bubbles. Inspect the channel under a microscope to ensure that no bubbles are present, and if bubbles are present, re-inject the ER1 solution.

I. Place the chip directly under the UV lamp, make sure the UV light device inside the hood is on and use the buttons to readjust the setting to "constant".

J. Treat with UV light for 20 minutes.

K. After UV treatment, gently aspirate ER1 from the channel through the same port until there is no more solution in the channel.

Wash both channels with 100 ul of ER2 solution, then with 200 ul of dPBS.

Part II: Coating

A. Prepare ECM according to the manufacturer’s instructions. If the manufacturer directs, it is recommended to aliquot and freeze ECM. Avoid multiple freeze-thaw cycles.

B. Calculate the total volume of ECM solution.

Minimum volume for channel

i. Upper: 50ul

ii. Lower part: 20ul

iii. ECM diluent: Customized for ECM and prepared on ice.

If using Matrigel, follow the Matrigel protocol (the Matrigel protocol makes sure to have "slush" ice, don't touch it, and make sure any heat destroys Matrigel).

C. Aspirate dPBS from the channel.

D. Load the channel with ECM solution.

i. Pipette 30 ul of cold ECM solution using the tip.

ii. Use a pipette tip to probe the injection port in the lower channel on the upper surface of the chip near the port.

iii. After finding the port, insert the tip vertically into the port (connect tightly).

iv. Inject 30 ul of ECM solution and keep pressing the pipette plunger (if you see the discharged fluid coming out, the injection was successful, if you see the fluid coming out of the same injection port, the tip was not injected properly and repeat step ii).

v. To remove the tip, gently press the chip body using sterile forceps to remove the tip.

vi. Aspirate excess fluid from the chip surface (avoiding contact with the ports).

vii. Repeat the same procedure for the upper channel using 50 ul of ECM solution.

E. Incubate overnight at 4°C or for 2 hours at 37°C.

F. Seal the dish containing the coated chips using parafilm.

Example 3

This example provides one embodiment of a protocol for seeding cells within a chip in the upper channel (which is oriented horizontally unless otherwise indicated). This example assumes aseptic technique and a sterile environment.

It should be noted that although some cells require very specific seeding conditions, in general, optimal seeding densities are achieved when cells are closely spaced in a flat monolayer. From this spacing, most primary cells will attach and spread into a confluent monolayer.

See “Gravity Wash” below. This involves a) placing a drop (bolus) of media (100uL) onto a port on one side of the channel, taking care not to introduce any air bubbles within the port itself, and b) flowing it through the chip, always aspirating excess media from the exhaust port.

A. Move the chip to the hood.

B. Place it in a sterile dish (e.g., a 15 mm culture dish).

C. Lightly wash the chips.

i. Pipette 200 ul of cell culture medium using the tip.

ii. Use a pipette tip to probe the injection port in the lower channel on the upper surface of the chip near the port.

iii. After finding the port, insert the tip vertically into the port (connect firmly).

iv. Wash 200 ul of medium, continuing to press the pipette plunger (if you see fluid coming out, the wash was successful; if you see fluid coming out of the same injection port, the tip was not injected properly and repeat step iv).

v. To remove the tip, gently press the chip body using sterile forceps to remove the tip, while continuing to press the pipette plunger.

vi. Aspirate the discharged fluid.

vii. Repeat the same procedure to clean the upper channel.

viii. Repeat the washing step once more for both channels.

ix. Add media drops to the inlet and outlet ports (100 ul each).

D. Cover the dish and place in the incubator until the cells are ready.

E. Prepare a cell suspension and count the number of cells.

F. Seeding density is specific to the upper and lower channels, cell type, and user-defined requirements.

i. Upper channel: For example, Caco2 cells: 2.5 million cells/ml

ii. Lower channel: For example, HUVEC: Front growth

G. After counting the cells, adjust the cell suspension to an appropriate density.

H. For upper channel seeding, place the dish containing the chips in the hood and aspirate excess media on the chip surface (handle the chip carriers by their wings only, keep the chip carriers flat and do not pick them up! This will ensure even distribution of cells across the chip culture membrane).

I. Gently aspirate the cell suspension before seeding each chip.

J. Pipette 50 μl of cell suspension and seed into the upper channel (when the chip is in horizontal position, the upper channel is the lower right port) (use one chip first).

i. Place the pipette in a completely vertical position and insert it into the upper channel (verticality allows for a smooth introduction into the chip and ensures a more even cell distribution).

ii. Inject 50 ul of cell suspension and keep pressing the pipette plunger (if you see the discharged fluid coming out, the injection was successful, if you see the fluid coming out of the same injection port, the tip was not injected properly and repeat step ii).

iii. To remove the pipette, gently press the chip body using sterile forceps, excluding the cell culture area, to remove the tip, and continue to press the plunger.

iv. Using the seeded tip, immediately aspirate the discharge fluid from the chip surface (avoid contact with the port).

v. Use a pipette with a seeded tip to immediately remove any spillage from the chip surface.

Remove effluent from both the inlet and outlet to ensure they are flush with the chip surface and prevent hydrostatic flow.

K. Cover the dish and transfer it to a microscope to check the density.

L. After seeding, the chip is placed in an incubator until cells attach.

i. Place a small reservoir (15 ml or 50 ml conical tube cap) with PBS inside the dish to provide humidity to the cells.

ii. The attachment time ranges from 1 to 3 hours depending on the cell type.

M. After cells are attached, the chip is gravity washed with warm medium by gently washing the medium through the channel.

N. Place the chip back in the incubator until ready to move on to the next step.

Example 4

This example provides one embodiment of a protocol for seeding cells within a chip in the lower channel (which is oriented horizontally unless otherwise indicated). This example assumes aseptic technique and a sterile environment.

It should be noted that although some cells require very specific seeding conditions, in general, optimal seeding densities are achieved when cells are closely spaced in a flat monolayer. From this spacing, most primary cells will attach and spread into a confluent monolayer.

See “Gravity Wash” below. This involves a) placing a drop (bolus) of media (100uL) onto a port on one side of the channel, taking care not to introduce any air bubbles within the port itself, and b) flowing it through the chip, always aspirating excess media from the exhaust port.

A. Place the dish containing the chips into the hood and aspirate excess media on the chip surface (handle the chip carriers by their wings only, keep the chip carriers flat and do not pick them up! This will ensure even distribution of cells across the chip culture membrane).

B. Gently aspirate the cell suspension before seeding each chip.

C. Pipette 20 μL of cell suspension and seed into the lower channel (when the chip is in horizontal position, the lower channel is the upper right port) (use one chip first).

i. Inject 20 ul of cell suspension and keep pressing the pipette plunger (if you see the discharged fluid coming out, the injection was successful, if you see the fluid coming out of the same injection port, the tip was not injected properly and repeat step ii).

ii. To remove the pipette tip, gently press the chip body using sterile forceps, excluding the cell culture area, to remove the tip, and continue to press the plunger.

iii. Immediately aspirate the discharge fluid from the chip surface using the seeded tip (avoiding contact with the port).

iv. Remove the effluent by making both the inlet and outlet flush with the chip surface to prevent hydrostatic flow.

D. Cover the dish and transfer it to a microscope to check the density.

E. After seeding, flip the chip inside the dish and place the chip in the incubator until the cells attach to the underside of the membrane.

i. Attachment time ranges from 1 to 3 hours depending on cell type.

ii. Place a small reservoir (15 ml or 50 ml conical tube cap) with PBS inside the dish to provide humidity to the cells.

F. After cells are attached, the chip is flipped over again and gravity washed with warm medium by gently pouring medium through the channels.

G. Place the chip back in the incubator until ready to move to the next step (cells can be cultured on the chip under static conditions until ready to be connected to a perfusion manifold for flow conditions).

i. Suction off old badges from the chip surface.

ii. Gravity-wash the chip with warm media by gently injecting media through the channels daily, and drop media from the injection ports, 200 ul each for the upper and lower channels.

iii. Place a small reservoir (15 ml or 50 ml conical tube cap) with PBS inside the dish to provide humidity to the cells.

Example 5

In this example, one embodiment of a protocol for manufacturing a perfusion disposable or “pod” is provided. This assumes aseptic technique and a sterile environment.

A. Preheat the badge to 37℃.

B. Move the warm badge to the biohood.

C. Aliquot the required amount +5% into a 50 mL conical tube.

D. Sterilize one SteriFlip vacuum filter for each tube of the badge and transfer to the hood.

i. Remove the SteriFlip from the packaging and connect it to a 50 mL media tube.

ii. Connect to vacuum inside the hood and turn over.

iii. Vacuum degas for at least 15 minutes using a timer.

E. Prepare the correct number of PODs (based on the number of executable chips).

F. Sterilize the emulsion nest and trays with ethanol and move them to the hood.

G. For each viable chip, sterilize one packaged Pod with ethanol and transfer to the hood (always hold only the edges of the POD with your thumb and middle finger, keep the POD cover on top, and flatten the POD with your index finger while simultaneously holding it).

H. Remove the reservoir cover and add the media. This should produce a droplet suitable for drop-to-drop interlocking of the POD and chip.

i. Injection reservoir: Fill with 1-3 ml (minimum 1 ml).

ii. Outlet storage: 300 ul

I. Transfer the seeded chips from the incubator and bring them into the hood.

i. Remove the pipette tips using a gentle twisting motion and dispose of them.

ii. Using a 200 μl pipette, add 10-50 μl of media onto each port (avoiding bubbles forming inside the port). This should produce a droplet suitable for drop-to-drop engagement of the POD and chip.

J. Connect the chip+carrier to the POD. This connection process must create a drop-to-drop interlocking of the POD and chip using the drops formed in steps H and I.

i. With one hand, hold the chip carrier with the index finger and thumb so that the thumb is on the locking mechanism, and firmly hold the carrier.

ii. With the other hand, hold the Pod with your thumb and middle finger around the reservoir, and place your index finger on the top of the cover to secure it in place.

iii. Orient the Pod along its internal track so that you can see "inside".

iv. While still holding the carrier, align the feet of the carrier along the tracks inside the Pod.

v. Slide the chip carrier into the Pod.

vi. Using your thumb on the chip carrier, gently press the locking mechanism until it slides into place, capturing the chip inside the Pod.

vii. Ensure that each storage lid is correctly positioned on each Pod.

Claims (14)

a) a culture module comprising an actuating assembly configured to move a pressure manifold in relation to a plurality of microfluidic devices, said pressure manifold comprising an integrated valve;
b) The plurality of microfluidic devices are detachably connected to and in contact with the pressure manifold.
System. A system in accordance with claim 1, wherein the plurality of microfluidic devices are a plurality of perfusion disposable products. A system according to claim 1 or 2, wherein the integrated valve comprises a Schrader valve. In claim 1 or 2, each of the microfluidic devices further comprises a cover assembly comprising a cover having a plurality of ports, wherein the pressure manifold comprises a bonding surface having pressure points corresponding to the ports on the cover,
A system wherein said pressure points on the joint surface of the pressure manifold are in contact with said plurality of ports of the cover assembly. A system in accordance with claim 4, wherein the plurality of ports include a plurality of through-hole ports. A system in accordance with claim 4, wherein the plurality of ports are coupled with corresponding holes in the filter and the gasket. A system according to claim 4, wherein the culture module further comprises a pressure regulator. A system in accordance with claim 7, wherein the pressure regulator is configured to apply pressure through the pressure point. A system according to claim 1 or 2, wherein the operating assembly comprises a pneumatic cylinder operably connected to the pressure manifold. A system in accordance with claim 4, further comprising alignment features configured to align the bonding surface with each of the microfluidic devices. A system in accordance with claim 4, wherein the culture module further comprises an elastomeric membrane, and the elastomeric membrane is in contact with the microfluidic device. A system according to claim 1 or 2, wherein the culture module is configured to accommodate one or more trays, each tray including a plurality of microfluidic devices. A system according to claim 1 or 2, wherein the culture module further comprises a user interface for controlling the culture module. A system according to claim 1 or 2, wherein the plurality of microfluidic devices include ports configured to be aligned with respect to the integrated valve.

Images